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Characterization of the lung Krüppel-like transcription factor gene and upstream regulatory elements
Gene 236 (1999) 185–195 www.elsevier.com/locate/gene
Characterization of the lung Kru¨ppel-like transcription factor gene and upstream regulatory elements Jeffrey J. Schrick, Michael J. Hughes, Kathleen P. Anderson, Michelle L. Croyle, Jerry B. Lingrel * Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH 45267-0524, USA Received 2 March 1999; received in revised form 31 May 1999; accepted 7 June 1999; Received by A. Bernardi
Abstract We previously described the isolation and characterization of the cDNA for lung Kru¨ppel-like factor (LKLF ), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the −490/−72 bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from −157/−72 bp was found to be necessary for upregulating expression. In transfection assays, mutations of the −138/−111 bp region resulted in approximately 70–80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Gene regulation; LKLF; Transcription; Zinc finger
1. Introduction The formation of the mammalian lung is a complex process involving numerous precisely coordinated developmental events that give rise to the saccule, bronchi, bronchioles, and alveoli (Burri, 1991). Defects in this process are usually life-threatening, resulting in a high fetal mortality. The incidence of such defects in humans is largely unknown, due to the limited knowledge of genetic pathways in lung development. However, recent progress describing several important genes expressed in the lung has extended the understanding of the development of this critical organ. We have recently identified a transcription factor LKLF, whose expression in adult mice is limited largely Abbreviations: BKLF, Basic Kru¨ppel-like factors; CDX1, caudal-1; EKLF, erythroid; ES, embryonic stem cell; GKLF, gut; IKLF, intestine; LKLF, lung; PCR, polymerase chain reaction; RAG, renal adenocarcinoma cell. * Corresponding author. Tel.: +1-513-558-5324; fax: +1-513-558-8474. E-mail address: [email protected] (J.B. Lingrel )
to the lung (Anderson et al., 1995). LKLF is a member of a small group of transcription factors belonging to the Kru¨ppel-like family, which includes BKLF (Miller and Bieker, 1993), GKLF (Perkins et al., 1995), IKLF (Conkright et al., 1999), and EKLF (Crossley et al., 1995). EKLF, the erythroid Kru¨ppel-like factor, was the first lineage specific gene of this family to be characterized and is required for b-globin gene expression. It contains a zinc-finger domain that binds to a CACCC element in the promoter of the b-globin gene. All members of this family share an extensive carboxylterminal Kru¨ppel-like homology, but differ in the complexity and specificity of expression in adult tissue and during development. BKLF is the most widely expressed Kru¨ppel-like factor whereas GKLF, IKLF, and EKLF are more restricted to the gut, intestine and erythroid cells, respectively. Mice deficient in LKLF die in utero between 12.5 and 14.5 dpc ( Wani et al., 1998, Kuo et al., 1997a). Ablation of the LKLF gene results in abnormal blood vessel formation and stabilization during early murine embryogenesis. The specific defect is thought to be in
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late-stage blood vessel formation in recruitment of the pericytes and vascular smooth muscle cells for the normal vascular tunica media ( Kuo et al., 1997a). In addition, LKLF has also been shown to be required for transcriptional regulation of single positive maintenance of T-cell quiescence and survival ( Kuo et al., 1997b). Nevertheless, it is the blood vessel defects that contribute to the embryonic mortality in LKLF deficient mice. LKLF and its developmental role in the murine lung have yet to be described. LKLF is largely expressed in the lung and spleen of adults (Anderson et al., 1995) and is expressed in the early developing umbilical artery and vein ( Kuo et al., 1997b). LKLF is also expressed in resting T lymphocytes and the absence of its expression leads to the apoptotic death of these cells ( Kuo et al., 1997b). Moreover, in-situ hybridization studies have shown that LKLF is expressed in bronchial epithelium and type II cells in adult mouse lung (Maqsood Wani, pers. commun.). As a result, LKLF is also thought to play a role in lung development. The LKLF mutant mice die at 12 days of embryonic development, and therefore, a defect in this tissue cannot be seen as the lung is just beginning to develop at this time. ES cell chimera studies with lung explant cultures have demonstrated a requirement for LKLF in lung development ( Wani, unpublished results). To better understand the regulation of LKLF, we have isolated and sequenced the complete mouse LKLF gene and initiated a promoter characterization study for the cis-regulatory regions responsible for LKLF expression. We demonstrate that two SP1 sites contribute to the general expression levels in LA-4 lung cells and that a 30 bp region of the proximal LKLF promoter greatly influences reporter expression in these cell lines. It is further shown that unknown nuclear proteins bind to this region.
2. Materials and methods 2.1. Northern analysis
2.2. Sequencing, deletion constructs and site directed mutagenesis Sequencing of a 3.4 kb EcoRI LKLF genomic fragment was performed on an Applied Biosystems Inc. ( Foster City, CA) model 373 DNA sequenator using the Taq DyeDeoxy kit. Oligonucleotides used as primers for sequencing, PCR and electrophoretic mobility shift assay ( EMSA) were synthesized on an Applied Biosystems Inc. model 380B DNA synthesizer and were column- or gel-purified using PAGE. Sequence analysis was performed using DNanalyze. Transcription factor databases TRANSFAC (Collins, and Driesel, 1991) and TFSEARCH were used to search for putative transcription factor binding sites. A 1.9 kb Acc65I–SalI genomic fragment (−2017 to −72 of upstream promoter) was ligated to a 0.1 kb SalI–NcoI PCR fragment (−72 to +14) where the NcoI site had been mutated into a HindIII site. This construct was subcloned into the pGL3-basic luciferase reporter vector (Promega, Madison, WI ). Deletions in the promoter from −2017, −1643, −490, −243, −202, −157 and −72 to +14 were made either by restriction digestion or PCR. All PCR reactions were carried out in a Perkin-Elmer Cetus DNA Thermal Cycler. One hundred milliliter reactions used 0.2 mM of each primer and 30 pM template. Reaction conditions consisted of 35 cycles of 94°C for 1 min denaturing, 55°C for 1 min annealing and 72°C for 1 min extension. A final 72°C extension for 10 min was included at the end. Mutagenesis was performed by a modified overlap extension protocol (Ho et al., 1989). The list of the outer A and D primers as well as a list of all the mutagenic C, M1, M2 and M3 primers are shown in Section 2.5. The A primer was synthesized with an Acc65I site at the 5∞ end, and the D primer was synthesized with a HindIII site at the 3∞ end. The end product was then digested with Acc65I and HindIII, purified by agarose gel electrophoresis, extracted from the gel by QIAEX II (QIAGEN ) and ligated into pGL3-basic luciferase expression vector. All clones were sequenced to verify that only the desired mutations had been created. 2.3. Cell culture, transfections and reporter gene assays
Poly A+ RNA was isolated from cells using the Mini RiboSeptm Ultra mRNA Isolation Kit (Collaborative Biomedical Products, Bedford, MA). Northern blots were performed by the glyoxal denaturation method (Sambrook et al., 1989). Poly A+ RNA was fractionated on agarose gels, transferred to Sure Blot nylon membranes (Oncor, Gaithersburg, MD). Radiolabled probes for hybridization were either a 3.4 kb EcoRI LKLF genomic fragment, including a 2 kb promoter region through the first zinc finger coding region, or the entire LKLF cDNA (Anderson et al., 1995).
All cells were purchased from ATCC and grown according to recommended conditions. For assays, cells were plated in six well plates (1×105 cells/35 mm well ) 12 h prior to DOSPER (Boehringer Mannheim) liposomal transfection. The transfection efficiency was monitored by co-transfection of 0.2 mg pRL-SV40 (Promega) plasmid. One microgram of promoter construct was transfected with 1.8 mg of pGL3-basic plasmid for 3 mg of total DNA transfected per well. For the dual luciferase assays, cells were harvested 48 h after transfection as
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per the manufacturer’s protocol (Promega). Assays were performed using 10 ml of cell lysate in 100 ml of assay buffer. Luminescence was detected on a Monolight 2010 (Analyical Luminescence Laboratory) luminometer. In each set of experiments, expression was normalized to pRL-SV40 controls. The highest expressing construct (−243 to +14) was arbitrarily set at 100, and all other expression is presented as a percentage of that value. 2.4. Nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from LA-4 and RAG cells by the method described in Dignam et al. (1983). Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). EMSA was performed by a modified method described by Fong and Emerson (1992). Double-stranded oligonucleotides were labeled with [c-32P]ATP using T4 polynucleotide kinase (GIBCO). The binding conditions were as follows: 2×104 cpm of labeled oligonucleotide and 10 mg of protein in 20 ml of binding buffer [100 mM Hepes, pH 7.8, 100 mM KCl, 60 mM MgCl , 1 mM EDTA, 2 2.5 mM dithiothreitol (DTT ), 1 mg of poly dIdC ] for 20 min at 20°C. The optimal binding conditions for the 30 bp fragment were as follows: 60 mM HEPES, pH 7.9, 250 mM KCl, 60% glycerol, 20 mM Tris, pH 7.9, 6 mM EDTA, 5 mM DTT, 1 mM PMSF, 10 mg/ml of Aprotinin, 10 mg/ml of Leupeptin, 3.5 mg/ml of Pepstatin and 1 mg of poly dIdC. Competition assays were performed by the addition of 100× unlabelled oligonucleotide competitor. Sp1 consensus oligonucleotide (d.s. 5∞-GAT CGA TCG GGG CGG GGC GAT C-3∞) was purchased from Stratagene (La Jolla, CA). Specific vs. non-specific competition was determined by the addition of non-specific oligonucleotides in a separate assay. Resolution of the DNA–protein complex was done on a non-denaturing polyacrylamide gel (39:1 acrylamide:bis-acrylamide) with 1× TBE, 0.05% NP-40, 5% glycerol, 1 mM EDTA and 0.5 mM DTT. Gels were dried under vacuum and exposed to a phosphorimager screen (Molecular Bioproducts). 2.5. Oligonucleotides used for mutatgenesis Oligonucleotides were used for 4 bp mutagenesis of the −142 to −106 region of the LKLF promoter. The complements of the corresponding oligonucleotides are not shown. (A) Oligonucleotide; 5∞-CCGGGTACCCCGCCACAGC-3∞, (D) Oligonucleotide; 5∞-CCCAAGCTTGGGCAGGGACTG-3∞. C oligonucleotides: (A) 5∞-GGGGTACCCCGCCACAGCCACCAGGCGCAGGCTTATATA-3∞,
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(B) 5∞-CCACCACCGCTGAACTTATATACC-3∞, (C ) 5∞-CACCGCCAGGTCCGTATACCCGG∞-3∞, (D) 5∞-GCCAGGCTTACGCGCCGCGGCTAA-3∞, ( E) 5∞-GGCTTATATATTATGGCTAAATTT-3∞, (F ) 5∞-TATATACCGCAATCAAATTTAGGC-3∞, (G) 5∞-TACCGCGGTGG GCTTAGGCTGAC-3∞, (H ) 5∞-GCGGCTAAATCCGAGCTGAGCCCG-3∞, (I ) 5∞-CTAAATTTAGCGACAGCCCGGAGC-3∞. Mutant oligonucleotides for the 30 bp core region were derived from the mutant construct sequence used in cell transfection assays. An EcoRI site replaces the wild-type sequence creating a mutant binding site: Wild-type: 5∞-AGGCTTATATACCGCGGCTAAATTTAGGCT-3∞, M1: 5∞-GGAATTCCGCACCGCGGCTAAATTTAGGCT-3∞, M2: 5∞-AGGCTT TATGGAATTCCGCAATTTAGGCT-3∞, M3: 5∞-AGGCTTATATACCGCGGCTAGGAATTCCGC-3∞.
3. Results 3.1. Cloning and sequence analysis of the LKLF gene The high degree of similarity between LKLF and EKLF in the zinc finger region was exploited in the cloning of the LKLF gene. Using the zinc finger region of the EKLF cDNA as a probe, several phage clones were recovered from a mouse 129 genomic library. Fragments spanning the gene were subcloned and the nucleotide sequence were determined. Comparison of this genomic sequence with the LKLF cDNA determined the location of three exons containing the complete coding sequence. Fig. 1A shows a schematic diagram of the LKLF gene structure along with a list for intron/exon boundaries (Fig. 1B). The entire sequence structure of the gene has been submitted to GenBank. This genomic organization has many features in common with the mouse EKLF gene (Anderson et al., 1995). Both genes are composed of three exons and have very short 5∞ untranslated regions. The splice junctions are generally conserved, particularly in the zinc finger region that spans exons 2 and 3. Additionally, the relative sizes of the corresponding exons and introns between EKLF and LKLF are similar. The tissue distribution of LKLF, with expression generally limited to the lung and spleen in adult animals, is of particular interest since few transcription factors expressed in the lung exhibit such a restricted pattern. As a first step in defining the cis-elements necessary for expression of LKLF, we examined the 5∞ promoter sequence of the mouse LKLF gene. As shown in Fig. 1, our genomic clone contains 2017 bp of 5∞ flanking sequence. The proximal promoter sequence is extremely
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these cells were selected for the transfection studies with the LKLF promoter/reporter constructs. The mouse RAG renal adenocarcinoma cell line was chosen as a negative control for non-specific expression. The murine RAG cell line expresses extremely low levels of LKLF when compared with LA-4 cells (Fig. 2B) and should not express our LKLF promoter specific constructs at high levels. 3.3. Deletion analysis of the −2017 bp LKLF promoter
Fig. 1. Sequence structure of the LKLF genomic region. (A) Schematic diagram of the LKLF gene structure is shown. (B) Annotations for the intron/exon boundaries are shown with sequence from the entire gene submitted to GenBank (Accession No. B254090). A 3.4 kb EcoIRI fragment from a mouse 129 library was isolated and characterized using the zinc finger region of EKLF and the LKLF cDNA as probes.
GC-rich, with a composition of approximately 70% GC in the first 350 bp. Further 5∞, we noted a region of approximately 200 bp of purine-pyrimidine repeats. Both of these features are absent from the EKLF promoter region. 3.2. Northern analysis for LKLF expression in cell lines We previously described the expression of LKLF in several tissues including the adult lung (Anderson et al., 1995). In order to identify promoter elements necessary for expression, we screened several lung cell lines for the presence of LKLF. The LA-4, LL/2 and H292 cell lines (Fig. 2A) showed differential levels of LKLF expression in Northern blots probed with LKLF cDNA. The mouse cell line LL/2 is a Lewis lung carcinoma and LA-4 is derived from a mouse lung adenoma. Among the cell lines tested, the highest level of LKLF expression was found in LA-4 cells. No detectable LKLF expression was observed in H292 cells, a human mucoepidermoid lung cell line. We estimate that the LA-4 and LL/2 cells express 20–25% of the amount of LKLF RNA that is produced in adult mouse lung ( Fig. 2A). Since LA-4 cells also exhibit many aspects of lung type II cells (Stoner et al., 1978) and type II cells express LKLF based on an in-situ analysis ( Wani, unpublished results),
Large deletions of the LKLF promoter sequence were created within the −2017 to +14 bp region, relative to the start of transcription. These deletion fragments were attached to a luciferase reporter gene in pGL3 (see Section 2). The constructs were then transfected into LA-4 and RAG cells, and the levels of luciferase expression were determined 24–48 h following transfection. Deletion of the region −2017/−1643 bp showed very little effect on luciferase reporter gene expression ( Fig. 2B). Further deletion to −490 bp increased expression in LA-4 cells to nearly double that of the −1643 bp construct. This suggested that the −1643/−490 bp region may contain negative cis-acting regulatory element(s). The deletion to −243 bp produces a fragment with the highest levels of expression when compared with all other deletion constructs. Finally, the expression from this promoter dropped to baseline levels when only 72 bp of 5∞ flanking sequence were included. These data indicate that major determinants of LKLF promoter specific transcription are located in the −243/−72 bp region when assayed in lung cell lines. Further characterization concentrated on this 172 bp region. The 172 bp fragment was subjected to a computerbased scan for consensus binding sites of characterized transcription factors (Collins and Driesel, 1991). Two Sp1 consensus binding sites were identified in this search. The first Sp1 binding site at −222 to −202 bp was designated Sp1A, and the second at −202 to −182 bp was designated as Sp1B (Fig. 3C ). Sp1 is a Kru¨ppel-like factor that binds to GC box promoter elements and is found in a wide variety of cells types ( Kadonaga et al., 1987). Although this factor is found ubiquitously, expression levels vary considerably, depending on the cell type (Pieler and Bellefroid, 1994). The position of the Sp1 binding site relative to other promoter elements has also been shown to affect tissue-specific gene expression (Gregory et al., 1996). Since these sites were the most prominent elements revealed by the computer analysis, we examined the Sp1 consensus binding sites first. 3.4. Nuclear binding assays and deletion analysis of the Sp1 sites. In order to determine whether the Sp1 consensus sites are capable of binding Sp1, an EMSA was performed using nuclear extract from LA-4 cells. Binding of crude
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Fig. 2. LKLF expression and deletion analysis in lung cell lines. (A) Northern blot containing 2 mg/lane Poly A+ RNA fractionated on 1% agarose gels was hybridized with a LKLF specific radiolabled probe. Expression was seen in LL/2, a mouse Lewis lung carcinoma and LA-4, a mouse lung adenoma cell lines. No expression was detected in H292, a human mucoepidermoid lung cell line. (B) LKLF expressions in lung tissue (intentionally underloaded), RAG and LA-4 cell lines were compared. RAG cells express LKLF at approximately 5% of LA-4 cell levels. Internal lanes from this Northern blot were deleted. A b-actin specific radiolabled probe was hybridized as a loading control in (A) and (B). (C ) LKLF promoter deletion constructs were used to identify cis- regulatory elements in LA-4 cells. Various promoter deletion constructs are shown on the left (see also Section 2). Relative levels of luciferase expression (as a percentage of the highest) are shown in a horizontal bar graph. The highest expressing construct −243 to +14 was arbitrarily placed at 100. The error bars represent the standard error and n>6 in all columns. RAG cells were used as negative LKLF expressing cell control for all constructs.
nuclear extract to the −243/−157 bp region produces several specific shifted fragments, as indicated by the arrows seen in Fig. 3. When unlabelled oligonucleotides corresponding to either Sp1A or Sp1B are added to the binding reaction mix, the complex is competed off. Nonspecific oligonucleotides have no effect. To determine whether the binding factor is indeed Sp1, a supershift assay with anti-Sp1 antibody was performed. Slower mobility complexes are evident in EMSAs with the presence of the Sp1 antibody indicating that Sp1 can
bind to at least one of the consensus sites Sp1A or Sp1B ( Fig. 3B). While Sp1 can bind to these sites, such findings do not address whether this binding is important in LKLF transcription. To determine whether these sites are involved in expression of the LKLF gene, these sites were removed and the resulting constructs assayed by transfection. Loss of the first Sp1 site by deletion to −202 bp reduces expression levels in LA-4 cells to 76.6% (SE+5.18) of −243 bp construct expression levels,
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Fig. 3. Upstream promoter characterization with LA-4 nuclear extract and Sp1 antibody. (A) The binding assay was performed using LA-4 nuclear extract and the −243 to −157 bp region of the promoter. The first and second lane represent free probe and probe bound to extract, respectively. Subsequent lanes are competed with 100× molar excess cold oligonucleotide. (B) Sp1 antibody supershift of the upstream region of the LKLF promoter. The first lane represents probe and LA-4 nuclear extract, and the second lane contains probe and rabbit pre-immune serum. The promoter fragment, LA-4 nuclear extract, and rabbit Sp1 Ab are shown in lane three. The arrow indicates the supershifted complex of the Sp1 nuclear factor. Note, this gel has been run longer to resolve the supershifted band. (C ) The sequence below is the LKLF promoter from −243 to +14. The TATA box and HindIII insertion site have been boxed to show the relative positions. The sequence corresponding to Sp1A is underlined in bold, and the sequence corresponding to Sp1B is double-underlined.
whereas the −157 bp deletion reduces expression levels in LA-4 cells to 65.8% (SE+4.88) of −243 bp levels. Thus, the Sp1 sites contribute modestly to expression levels but are not absolutely required for reporter expression. Moreover, a reduction in expression to base line levels is seen in RAG cells when the Sp1 sites are removed ( Fig. 2B, −243 construct).
3.5. Analysis of the −157 to −72 promoter region Since the Sp1 elements appear to play only a modest role, we further examined the region between −157/−72 bp for transcriptional elements. A TRANSFAC and TFSEARCH database analysis of this region did not reveal a strong consensus binding site
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Fig. 4. Four base pair mutagenesis of the LKLF promoter from −142 to −106 bp: a graphic representation showing transfected reporter constructs containing the LKLF promoter from −142 to −106. Regions M1, M2, and M3 are in bold and underlined and shown as wild-type ( W.T.) sequence. The mutations are in bold and underlined lower-case sequence beside the graph (A–I ). Error bars represent the standard error and n>6 in all columns. Black bars represent LA-4 cell, and gray bars represent RAG cell transfections.
that might confer lung gene expression. However, using the TFSEARCH website, two consensus binding sites in the −157/−72 bp fragment showed a sequence similarity with an element in the promoter for CDX1 and for TATA-binding proteins. Oligomers designed from the CDX1 sequence failed to compete the bound −157/−72 bp fragment from mouse lung nuclear extracts (data not shown). Moreover, hTAF p130 antibodies (for TATA-complex proteins) did not supershift lung extracts in similar EMSAs (data not shown). Finally, the human LKLF upstream sequence was compared with the murine sequence in Fig. 1 (data not shown, Wani et al., 1999). This identified a highly conserved sequence homology within the –157/−72 bp region. With no obvious candidate sequences, linker scanning mutagenesis was employed to search for potential functionally important elements. The 86 bp upstream region was mutated to produce a series of small replacement constructs within the −157 bp deletion. Mutational analysis of the −142/−106 bp region showed the greatest effect on expression in La-4 cells (data not shown). To further localize the cis-elements responsible for expression, regions M1, M2 and M3 were mutated in 4 bp increments using a 4 bp replacement strategy (Fig. 4). This analysis revealed separate regions responsible for reporter expression. The −138/−127 bp and
−122/−111 bp regions are critical, whereas mutation within the −126/−123 bp region has a more modest affect on gene expression ( Fig. 4). In addition, the TATA box of the LKLF promoter was identified by mutating an A to G and a T to C. In the context of the larger promoter (−243/+14), this mutation reduces the expression to 17.5% (SE+0.61) of the −243 reporter construct, indicating that the TATA box is required for LKLF transcription (data not shown). To determine whether nuclear factors bind to these functionally important regions, EMSAs were performed on the −157/−72 bp LKLF fragment using nuclear extracts from LA-4 and LL/2 cells. Three specific bands were observed (Fig. 5). When competed with either the A oligonucleotide or the E oligonucleotide, corresponding to the two sites critical for reporter expression (−138/−127 bp and −122/−111 bp), the two lower bands as well as a faint upper band disappear. Thus, specific competition with both the A and the E oligonucleotides can disrupt binding of nuclear factors to the −157/−72 bp region of the LKLF promoter. 3.6. Analysis of a core 30 bp promoter region Since the combined linker scanning and EMSA data suggest that an approximate 30 bp region is important for transcription, we investigated this region in more
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Fig. 5. Electrophoretic mobility shift assay of the −157/ −72 region of the LKLF promoter. Binding assays done on the −157/ −72 region of the LKLF promoter reveal a characteristic banding pattern (see arrows) that is disrupted when competed with 100× molar cold oligonucleotide A (−142 to −128 bp) or E (−125 to –108 bp). Binding with non-specific oligonucleotides shows no disruption in binding pattern (data not shown). Regions A and E correspond to the dark line and label in the sequence below the gel shift assay. Nuclear extract was prepared from LA-4 cells and LL/2 cells. A 5% non-denaturing polyacrylamide gel was run at 250 V for 2 h at 4°C
detail. We sought to determine whether the functional data from the LA-4 cell transfections would be reflected by a similar EMSA shift using adult lung tissue nuclear extracts. Under optimal binding conditions for this 30 bp fragment, a strong single specific band was seen with crude nuclear extract from adult lung ( Fig. 6A). This EMSA band showed a higher percentage of bound fragment (approximately 50%) than with the larger −157/−72 bp fragment and LA-4 nuclear extract (Fig. 5). A DNase I footprint analysis of this region with either the −157/−72 fragment or 30 bp region failed to show any binding residues. Furthermore, modifications using a methylation interference footprint on the 30 bp fragment also yielded no localization of a core binding sequence. To further determine the specificity of the protein complex binding to the 30 bp fragment, we mutated
oligonucleotides in three different regions of the −139/−110 bp binding sequence. Fig. 6A shows EMSAs containing oligonucleotides where the wild-type ( WT ) sequence is replaced with an EcoRI site (see Section 2.5). The M1 mutant oligonucleotide changes the 5∞ sequence (see Section 2.5) and causes a faster mobility complex shift of the protein–DNA interaction. The M2 and M3 mutant oligonucleotides change the middle 10 bases and the 3∞ sequence of the 30 bp fragment (see Section 2.5). No significant binding was seen with the M2 and M3 mutant oligonucleotides. These results suggest that the middle and 3∞ sequences are critical for overall binding and that the 5∞ region potentially binds a subset of proteins that bind to the wild-type fragment. Our linker-scanning cell expression functional data suggest that this 30 bp region is crucial for LKLF promoter expression in the LA-4 lung cell line. Moreover, the molecular binding of this 30 bp region is disrupted by site-specific mutagenesis. To determine whether the binding is specific for adult mouse nuclear extracts, we screened specific tissues that do and do not express LKLF using the wild-type 30 bp fragment. Fig. 6B shows EMSAs using adult brain, kidney, liver and lung nuclear extracts bound to the WT fragment. Extracts from liver and brain exhibit a similar mobility shift as seen with the lung extract. However, a lower mobility shift band is seen with kidney extract using the WT oligonucleotide. Interestingly, this lower shifted band is similar in size to the M1 mutant oligonucleotide ( Fig. 6A). Finally, to determine whether indeed a complex is formed in a differential manner, the WT and M1 oligouncleotides were bound with adult brain, kidney, liver and lung nuclear extract (Fig. 6B). All extracts with the M1 oligonucleotide appear to bind the same lower shifted band, as seen in kidney with the WT fragment. AP1 and SP1 oligonucleotide controls demonstrated that protein extracts were not degraded (data not shown). Collectively, this suggests that a protein(s) binding to the 5∞ most region of the 30 bp fragment has a differential expression pattern and appears to bind in a functionally important region.
4. Discussion The mouse LKLF gene is structurally related to the EKLF gene. There is conservation in the number of exons and the splice junctions. The 5∞ promoter regions vary considerably, however, as anticipated from the different expression patterns observed with these two transcription factors. In this study, it is shown that LKLF is expressed in lung tissue as well as in a number of cell lines derived from lung. In addition to this expression, we have observed comparable levels of LKLF expression in macrophage, endothelial and
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Fig. 6. (A) Electrophoretic mobility shift assay of the 30 bp (− 139/−110 bp) region of the LKLF promoter. Binding assays were done on the −139/−110 bp region of the LKLF promoter. A single shifted fragment (black arrow) was seen using the wild-type ( W ) sequence as labeled oligonucleotide and was competed away using cold fragment as before. The mutant M1 oligonucleotide shifted to a lower molecular weight fragment (gray arrow) than the W probe. Cold M1 marginally competes for the M1 probe, whereas W competes for nearly all the bound fragment. Mutant M2 and M3 probes failed to show significant binding. S, self-oligouncleotide; W, wild-type oligonucleotide. Adult lung nuclear extract was used. Bar schematic shows the position of the mutated oligonucleotides. (B) Tissue distribution EMSA of the 30 bp LKLF promoter fragment. Binding assays were done using a wild-type and mutant −139/−110 bp region of the LKLF promoter. Different tissues from nuclear extracts were tested with the wild-type ( W ) and mutant M1 probes. N, no extract; B, brain; K, kidney; Li, liver; Lu, lung. As in Fig. 6A, brain, liver and lung extract showed a similar band shift with the W probe (black arrow). However, a lower molecular fragment was seen with the kidney extract (gray arrow) using the W probe. All tissues tested showed the lower molecular weight shift with the M1 oligonucleotide probe (gray arrow). Competition oligonucleotides were as before (data not shown).
embryonic stem cell lines (data not shown). Our major interest is to describe the upstream regulators for LKLF expression and to identify specific cis-regulatory elements. The LA-4 murine lung cell line was chosen for analysis of LKLF promoter expression because it represents type II lung cells that express LKLF. Approximately 2 kb of the LKLF promoter were analyzed by deletion and linker scanning analysis. We observed one region with a repressive effect on reporter expression and one region of transcriptional activation. Although there is very little change between the −2017 and −1643 promoter deletion constructs, there is a twofold difference between the −1643 and −490 con-
structs. Within this 1153 bp segment are large regions of repeats and several transcription factor consensus binding sites. Interestingly, expression of the LKLF protein is able to overcome the negative regulation and restore the −2017 promoter construct to approximately −490 construct levels (data not shown). This activity could be derived from the binding of LKLF directly to the promoter and the activation of transcription in a manner consistent with other Kru¨ppel-like factors. Alternatively, LKLF could interact with negative transfactors to derepress expression. Precedence for such an interaction comes from studies demonstrating that some zinc finger proteins are able to associate with other
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transcription factors via the zinc finger domains (Crossley et al., 1995; Merika and Orkin, 1995; Gregory et al., 1996). The deletion of the putative Sp1 elements in the LKLF promoter results in a decrease in reporter gene expression. Sp1, also a Kru¨ppel-like factor that binds to GC box promoter elements ( Kadonaga et al., 1987), is found ubiquitously, but expression levels can vary 1000-fold, depending on the cell type (Pieler and Bellefroid, 1994). Tissue-specific gene expression can also be affected, depending on the Sp1 binding site position relative to other promoter elements (Gregory et al., 1996). In our study, deletion of the Sp1 elements reduces overall LKLF promoter activity but does not eliminate it. We conclude that Sp1 may be involved in the level of LKLF expression, but it is not critical for lung cell gene expression. Moreover, deletion of the Sp1 sites also reduces expression in RAG cells using the larger constructs. Therefore, the functional data indicate that these sites only contribute a modest activation as basal transcription elements. Mutations in the remaining sequence from −157/−72 produce different results when compared between LA-4 and RAG cells, indicating that this region is likely responsible for gene regulation. Linker scanning analysis of this region demonstrates that it is important for cell-line expression. Binding of LA-4 nuclear extracts confirms that proteins bind to this region. Attempts at obtaining a DNase I footprint in this region were unsuccessful, perhaps due to a low binding affinity of the larger fragment, the concentration of binding factor in the crude nuclear extract, or a labile protein complex. Mutational analysis, however, has shown that at least two regions are crucial for gene expression. Thus, when either one of the regions is mutated, the promoter is unable to direct cell-line expression of the reporter gene. The EMSA data correspond with the mutation studies in that competition with oligonucleotides derived from either of these regions abolishes specific binding to the entire −157/−72 probe. It is possible that one protein binds to the promoter region from −139/−110 and that −126/−123 are not essential for binding. This would also be consistent with the fact that the entire complex is disrupted when competed with either one or the other oligonucleotide. Another possibility is that two or more proteins bind to these regions, and binding of one requires binding of the other(s). The doublet pattern seen in gel shifts of longer run gels ( Fig. 5) could be indicative of more than one protein in the complex, or this could be the result of a modification such as phosphorylation of one or more of the binding proteins. Finally, using the 30 bp (−139/−110 bp) region, it is shown that mutated oligonucleotides cannot bind nuclear extract (Fig. 6A). The 5∞ region of this core element is not essential for binding as the remaining
wild-type 20 base sequence continues to bind protein(s). However, no protein binds to the mid- and 3∞ sequence when it is altered, suggesting that these regions are critical for binding of the protein complex overall. Base alterations of the 30 bp fragment, using DMS and DEPC footprinting methods, disrupted overall binding of extract to the probe. The base composition and sensitivity of this cis-regulatory region might also explain why our attempts to footprint the site failed. Critical nucleotides are likely altered so that no bound fragment can be obtained. Since several loosely conserved proteins that might bind to the −139/−110 region were ruled out, it is possible that a novel transcription factor binds to this site. In this study, a 2 kb promoter fragment 5∞ of the LKLF gene has been characterized. The upstream sequence includes a region from −157/−72 bp that is responsible for upregulating expression in LA-4 lung cell-line. Mutations within a 30 bp cis-element disrupt nuclear protein binding. Moreover, nuclear extracts from various tissues bind to this element in a differential manner. Although none of the cis-elements identified thus far exclusively binds to lung nuclear extracts, several functionally important regions do contribute to higher levels of reporter gene expression in the LA-4 lung cell lines. Identification of the factor(s) that bind the −139/−110 bp fragment awaits further isolation and characterization.
Acknowledgements This work was supported by NIH grants, HL57281 and POEMB HL41496. We thank Michael Conkright for the Northern blot data and helpful discussions from other members of the Lingrel laboratory.
References Anderson, K.P., Kern, C.B., Crable, S.C., Lingrel, J.B., 1995. Isolation of a gene encoding a functional zinc finger protein homologous to erythroid Kru¨ppel-like factor: identification of a new multigene family. Mol. Cell. Biol. 15, 5957–5965. Burri, P.H., 1991. Postnatal development and growth. In: Crystal, G., West, J.B. ( Eds.), The Lung: Scientific Foundations. Raven Press, New York, pp. 677–686. Collins, J., Driesel, A.J., 1991. Biotech Forum. Adv. Mol. Genet. 4, 95–108. Conkright, M.D., Wani, M.A., Anderson, K.P., Lingrel, J.B., 1999. A gene encoding an intestional-enriched member of the Kru¨ppel-Like factor family expressed in intestinal epithelial cells. Nucleic Acids Res. 27, 1263–1270. Crossley, M., Merika, M., Orkin, S.H., 1995. Self-association of the erythroid transcription factor GATA-1 mediated by its zinc finger domains. Mol. Cell. Biol. 15, 2448–2456. Dignam, J.D., Lebovitz, R.M., Roeder, R.G., 1983. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.
J.J. Schrick et al. / Gene 236 (1999) 185–195 Fong, T.C., Emerson, B.M., 1992. The erythroid-specific protein cGATA-1 mediates distal enhancer activity through a specialized beta-globin TATA box. Genes Dev. 6, 521–532. Gregory, R.C., Taxman, D.J., Seshasayee, D., Kensinger, M.H., Bieker, J.J., Wojchowski, D.M., 1996. Functional interaction of GATA1 with erythroid Kru¨ppel-like factor and Sp1 at defined erythroid promoters. Blood 87, 1793–1801. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K., Pease, L.R., 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59. Kadonaga, J.T., Carner, K.R., Masiarz, F.R., Tjian, R., 1987. Isolation of cDNA transcription factor Sp1 and functional analysis of the DNA binding domain. Cell 51, 1079–1090. Kuo, C.T., Veselits, M.L., Barton, K.P., Lu, M.M., Clendenin, C., Leiden, J.M., 1997a. The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis. Genes Dev. 11, 2996–3006. Kuo, C.T., Veselits, M.L., Leiden, J.M., 1997b. LKLF: A transcriptional regulator of single-positive T cell quiescence and survival. Science 277, 1968–1990. Merika, M., Orkin, S.H., 1995. Functional synergy and physical interactions of the erythroid transcription factor GATA-1 with the
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Kru¨ppel family proteins Sp1 and EKLF. Mol. Cell. Biol. 15, 2437–2447. Miller, I.J., Bieker, J.J., 1993. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kru¨ppel family of nuclear proteins. Mol. Cell. Biol. 13, 2776–2786. Perkins, A.C., Sharpe, A.H., Orkin, S.H., 1995. Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375, 318–322. Pieler, T., Bellefroid, E., 1994. Perspectives on zinc finger protein function and evolution — an update. Mol. Biol. Rep. 20, 1–8. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. Stoner, G.D., Hallman, M., Troxell, M.C., 1978. Lecithin biosynthesis in a clonal line of lung adenoma cells with type II alveolar cell properties. Exp. Mol. Path. 29, 102–114. Wani, M.A., Means, R.T., Lingrel, J.B., 1998. Loss of LKLF results in embryomic lethality in mice. Transgenic Res. 7, 229–238. Wani, M.A., Conkright, M.D., Jeffries, S., Hughes, M.J., Lingrel, J.B., 1999. cDNA isolation, genomic structure, regulation and chromosomal localization of human Lung Kruppel-like Factor, in press.
Characterization of the lung Kru¨ppel-like transcription factor gene and upstream regulatory elements Jeffrey J. Schrick, Michael J. Hughes, Kathleen P. Anderson, Michelle L. Croyle, Jerry B. Lingrel * Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, Cincinnati, OH 45267-0524, USA Received 2 March 1999; received in revised form 31 May 1999; accepted 7 June 1999; Received by A. Bernardi
Abstract We previously described the isolation and characterization of the cDNA for lung Kru¨ppel-like factor (LKLF ), a zinc finger transcription factor that is predominately expressed in the lung of adult mice. In this study, we report the complete structure and nucleotide sequence of the mouse LKLF gene, which is comprised of three exons and two small introns. Moreover, the identification of critical sequence elements required for expression is described using reporter constructs with the LKLF promoter transfected into LA-4 lung cells. Results from these constructs reveal an important region for transcriptional activity that lies between the −490/−72 bp upstream sequence. This region contains two canonical Sp1 binding sites that affect expression levels in a non tissue-specific manner. In addition, using a base-pair mutagenesis strategy, a region from −157/−72 bp was found to be necessary for upregulating expression. In transfection assays, mutations of the −138/−111 bp region resulted in approximately 70–80% loss of promoter activity. This cis-element does not appear to correspond to any known transcription factor consensus sequence. Moreover, mutations within this cis-region disrupt the binding of a protein complex from nuclear extracts of various tissues. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Gene regulation; LKLF; Transcription; Zinc finger
1. Introduction The formation of the mammalian lung is a complex process involving numerous precisely coordinated developmental events that give rise to the saccule, bronchi, bronchioles, and alveoli (Burri, 1991). Defects in this process are usually life-threatening, resulting in a high fetal mortality. The incidence of such defects in humans is largely unknown, due to the limited knowledge of genetic pathways in lung development. However, recent progress describing several important genes expressed in the lung has extended the understanding of the development of this critical organ. We have recently identified a transcription factor LKLF, whose expression in adult mice is limited largely Abbreviations: BKLF, Basic Kru¨ppel-like factors; CDX1, caudal-1; EKLF, erythroid; ES, embryonic stem cell; GKLF, gut; IKLF, intestine; LKLF, lung; PCR, polymerase chain reaction; RAG, renal adenocarcinoma cell. * Corresponding author. Tel.: +1-513-558-5324; fax: +1-513-558-8474. E-mail address: [email protected] (J.B. Lingrel )
to the lung (Anderson et al., 1995). LKLF is a member of a small group of transcription factors belonging to the Kru¨ppel-like family, which includes BKLF (Miller and Bieker, 1993), GKLF (Perkins et al., 1995), IKLF (Conkright et al., 1999), and EKLF (Crossley et al., 1995). EKLF, the erythroid Kru¨ppel-like factor, was the first lineage specific gene of this family to be characterized and is required for b-globin gene expression. It contains a zinc-finger domain that binds to a CACCC element in the promoter of the b-globin gene. All members of this family share an extensive carboxylterminal Kru¨ppel-like homology, but differ in the complexity and specificity of expression in adult tissue and during development. BKLF is the most widely expressed Kru¨ppel-like factor whereas GKLF, IKLF, and EKLF are more restricted to the gut, intestine and erythroid cells, respectively. Mice deficient in LKLF die in utero between 12.5 and 14.5 dpc ( Wani et al., 1998, Kuo et al., 1997a). Ablation of the LKLF gene results in abnormal blood vessel formation and stabilization during early murine embryogenesis. The specific defect is thought to be in
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late-stage blood vessel formation in recruitment of the pericytes and vascular smooth muscle cells for the normal vascular tunica media ( Kuo et al., 1997a). In addition, LKLF has also been shown to be required for transcriptional regulation of single positive maintenance of T-cell quiescence and survival ( Kuo et al., 1997b). Nevertheless, it is the blood vessel defects that contribute to the embryonic mortality in LKLF deficient mice. LKLF and its developmental role in the murine lung have yet to be described. LKLF is largely expressed in the lung and spleen of adults (Anderson et al., 1995) and is expressed in the early developing umbilical artery and vein ( Kuo et al., 1997b). LKLF is also expressed in resting T lymphocytes and the absence of its expression leads to the apoptotic death of these cells ( Kuo et al., 1997b). Moreover, in-situ hybridization studies have shown that LKLF is expressed in bronchial epithelium and type II cells in adult mouse lung (Maqsood Wani, pers. commun.). As a result, LKLF is also thought to play a role in lung development. The LKLF mutant mice die at 12 days of embryonic development, and therefore, a defect in this tissue cannot be seen as the lung is just beginning to develop at this time. ES cell chimera studies with lung explant cultures have demonstrated a requirement for LKLF in lung development ( Wani, unpublished results). To better understand the regulation of LKLF, we have isolated and sequenced the complete mouse LKLF gene and initiated a promoter characterization study for the cis-regulatory regions responsible for LKLF expression. We demonstrate that two SP1 sites contribute to the general expression levels in LA-4 lung cells and that a 30 bp region of the proximal LKLF promoter greatly influences reporter expression in these cell lines. It is further shown that unknown nuclear proteins bind to this region.
2. Materials and methods 2.1. Northern analysis
2.2. Sequencing, deletion constructs and site directed mutagenesis Sequencing of a 3.4 kb EcoRI LKLF genomic fragment was performed on an Applied Biosystems Inc. ( Foster City, CA) model 373 DNA sequenator using the Taq DyeDeoxy kit. Oligonucleotides used as primers for sequencing, PCR and electrophoretic mobility shift assay ( EMSA) were synthesized on an Applied Biosystems Inc. model 380B DNA synthesizer and were column- or gel-purified using PAGE. Sequence analysis was performed using DNanalyze. Transcription factor databases TRANSFAC (Collins, and Driesel, 1991) and TFSEARCH were used to search for putative transcription factor binding sites. A 1.9 kb Acc65I–SalI genomic fragment (−2017 to −72 of upstream promoter) was ligated to a 0.1 kb SalI–NcoI PCR fragment (−72 to +14) where the NcoI site had been mutated into a HindIII site. This construct was subcloned into the pGL3-basic luciferase reporter vector (Promega, Madison, WI ). Deletions in the promoter from −2017, −1643, −490, −243, −202, −157 and −72 to +14 were made either by restriction digestion or PCR. All PCR reactions were carried out in a Perkin-Elmer Cetus DNA Thermal Cycler. One hundred milliliter reactions used 0.2 mM of each primer and 30 pM template. Reaction conditions consisted of 35 cycles of 94°C for 1 min denaturing, 55°C for 1 min annealing and 72°C for 1 min extension. A final 72°C extension for 10 min was included at the end. Mutagenesis was performed by a modified overlap extension protocol (Ho et al., 1989). The list of the outer A and D primers as well as a list of all the mutagenic C, M1, M2 and M3 primers are shown in Section 2.5. The A primer was synthesized with an Acc65I site at the 5∞ end, and the D primer was synthesized with a HindIII site at the 3∞ end. The end product was then digested with Acc65I and HindIII, purified by agarose gel electrophoresis, extracted from the gel by QIAEX II (QIAGEN ) and ligated into pGL3-basic luciferase expression vector. All clones were sequenced to verify that only the desired mutations had been created. 2.3. Cell culture, transfections and reporter gene assays
Poly A+ RNA was isolated from cells using the Mini RiboSeptm Ultra mRNA Isolation Kit (Collaborative Biomedical Products, Bedford, MA). Northern blots were performed by the glyoxal denaturation method (Sambrook et al., 1989). Poly A+ RNA was fractionated on agarose gels, transferred to Sure Blot nylon membranes (Oncor, Gaithersburg, MD). Radiolabled probes for hybridization were either a 3.4 kb EcoRI LKLF genomic fragment, including a 2 kb promoter region through the first zinc finger coding region, or the entire LKLF cDNA (Anderson et al., 1995).
All cells were purchased from ATCC and grown according to recommended conditions. For assays, cells were plated in six well plates (1×105 cells/35 mm well ) 12 h prior to DOSPER (Boehringer Mannheim) liposomal transfection. The transfection efficiency was monitored by co-transfection of 0.2 mg pRL-SV40 (Promega) plasmid. One microgram of promoter construct was transfected with 1.8 mg of pGL3-basic plasmid for 3 mg of total DNA transfected per well. For the dual luciferase assays, cells were harvested 48 h after transfection as
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per the manufacturer’s protocol (Promega). Assays were performed using 10 ml of cell lysate in 100 ml of assay buffer. Luminescence was detected on a Monolight 2010 (Analyical Luminescence Laboratory) luminometer. In each set of experiments, expression was normalized to pRL-SV40 controls. The highest expressing construct (−243 to +14) was arbitrarily set at 100, and all other expression is presented as a percentage of that value. 2.4. Nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from LA-4 and RAG cells by the method described in Dignam et al. (1983). Protein concentration was determined by the BCA assay (Pierce, Rockford, IL). EMSA was performed by a modified method described by Fong and Emerson (1992). Double-stranded oligonucleotides were labeled with [c-32P]ATP using T4 polynucleotide kinase (GIBCO). The binding conditions were as follows: 2×104 cpm of labeled oligonucleotide and 10 mg of protein in 20 ml of binding buffer [100 mM Hepes, pH 7.8, 100 mM KCl, 60 mM MgCl , 1 mM EDTA, 2 2.5 mM dithiothreitol (DTT ), 1 mg of poly dIdC ] for 20 min at 20°C. The optimal binding conditions for the 30 bp fragment were as follows: 60 mM HEPES, pH 7.9, 250 mM KCl, 60% glycerol, 20 mM Tris, pH 7.9, 6 mM EDTA, 5 mM DTT, 1 mM PMSF, 10 mg/ml of Aprotinin, 10 mg/ml of Leupeptin, 3.5 mg/ml of Pepstatin and 1 mg of poly dIdC. Competition assays were performed by the addition of 100× unlabelled oligonucleotide competitor. Sp1 consensus oligonucleotide (d.s. 5∞-GAT CGA TCG GGG CGG GGC GAT C-3∞) was purchased from Stratagene (La Jolla, CA). Specific vs. non-specific competition was determined by the addition of non-specific oligonucleotides in a separate assay. Resolution of the DNA–protein complex was done on a non-denaturing polyacrylamide gel (39:1 acrylamide:bis-acrylamide) with 1× TBE, 0.05% NP-40, 5% glycerol, 1 mM EDTA and 0.5 mM DTT. Gels were dried under vacuum and exposed to a phosphorimager screen (Molecular Bioproducts). 2.5. Oligonucleotides used for mutatgenesis Oligonucleotides were used for 4 bp mutagenesis of the −142 to −106 region of the LKLF promoter. The complements of the corresponding oligonucleotides are not shown. (A) Oligonucleotide; 5∞-CCGGGTACCCCGCCACAGC-3∞, (D) Oligonucleotide; 5∞-CCCAAGCTTGGGCAGGGACTG-3∞. C oligonucleotides: (A) 5∞-GGGGTACCCCGCCACAGCCACCAGGCGCAGGCTTATATA-3∞,
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(B) 5∞-CCACCACCGCTGAACTTATATACC-3∞, (C ) 5∞-CACCGCCAGGTCCGTATACCCGG∞-3∞, (D) 5∞-GCCAGGCTTACGCGCCGCGGCTAA-3∞, ( E) 5∞-GGCTTATATATTATGGCTAAATTT-3∞, (F ) 5∞-TATATACCGCAATCAAATTTAGGC-3∞, (G) 5∞-TACCGCGGTGG GCTTAGGCTGAC-3∞, (H ) 5∞-GCGGCTAAATCCGAGCTGAGCCCG-3∞, (I ) 5∞-CTAAATTTAGCGACAGCCCGGAGC-3∞. Mutant oligonucleotides for the 30 bp core region were derived from the mutant construct sequence used in cell transfection assays. An EcoRI site replaces the wild-type sequence creating a mutant binding site: Wild-type: 5∞-AGGCTTATATACCGCGGCTAAATTTAGGCT-3∞, M1: 5∞-GGAATTCCGCACCGCGGCTAAATTTAGGCT-3∞, M2: 5∞-AGGCTT TATGGAATTCCGCAATTTAGGCT-3∞, M3: 5∞-AGGCTTATATACCGCGGCTAGGAATTCCGC-3∞.
3. Results 3.1. Cloning and sequence analysis of the LKLF gene The high degree of similarity between LKLF and EKLF in the zinc finger region was exploited in the cloning of the LKLF gene. Using the zinc finger region of the EKLF cDNA as a probe, several phage clones were recovered from a mouse 129 genomic library. Fragments spanning the gene were subcloned and the nucleotide sequence were determined. Comparison of this genomic sequence with the LKLF cDNA determined the location of three exons containing the complete coding sequence. Fig. 1A shows a schematic diagram of the LKLF gene structure along with a list for intron/exon boundaries (Fig. 1B). The entire sequence structure of the gene has been submitted to GenBank. This genomic organization has many features in common with the mouse EKLF gene (Anderson et al., 1995). Both genes are composed of three exons and have very short 5∞ untranslated regions. The splice junctions are generally conserved, particularly in the zinc finger region that spans exons 2 and 3. Additionally, the relative sizes of the corresponding exons and introns between EKLF and LKLF are similar. The tissue distribution of LKLF, with expression generally limited to the lung and spleen in adult animals, is of particular interest since few transcription factors expressed in the lung exhibit such a restricted pattern. As a first step in defining the cis-elements necessary for expression of LKLF, we examined the 5∞ promoter sequence of the mouse LKLF gene. As shown in Fig. 1, our genomic clone contains 2017 bp of 5∞ flanking sequence. The proximal promoter sequence is extremely
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these cells were selected for the transfection studies with the LKLF promoter/reporter constructs. The mouse RAG renal adenocarcinoma cell line was chosen as a negative control for non-specific expression. The murine RAG cell line expresses extremely low levels of LKLF when compared with LA-4 cells (Fig. 2B) and should not express our LKLF promoter specific constructs at high levels. 3.3. Deletion analysis of the −2017 bp LKLF promoter
Fig. 1. Sequence structure of the LKLF genomic region. (A) Schematic diagram of the LKLF gene structure is shown. (B) Annotations for the intron/exon boundaries are shown with sequence from the entire gene submitted to GenBank (Accession No. B254090). A 3.4 kb EcoIRI fragment from a mouse 129 library was isolated and characterized using the zinc finger region of EKLF and the LKLF cDNA as probes.
GC-rich, with a composition of approximately 70% GC in the first 350 bp. Further 5∞, we noted a region of approximately 200 bp of purine-pyrimidine repeats. Both of these features are absent from the EKLF promoter region. 3.2. Northern analysis for LKLF expression in cell lines We previously described the expression of LKLF in several tissues including the adult lung (Anderson et al., 1995). In order to identify promoter elements necessary for expression, we screened several lung cell lines for the presence of LKLF. The LA-4, LL/2 and H292 cell lines (Fig. 2A) showed differential levels of LKLF expression in Northern blots probed with LKLF cDNA. The mouse cell line LL/2 is a Lewis lung carcinoma and LA-4 is derived from a mouse lung adenoma. Among the cell lines tested, the highest level of LKLF expression was found in LA-4 cells. No detectable LKLF expression was observed in H292 cells, a human mucoepidermoid lung cell line. We estimate that the LA-4 and LL/2 cells express 20–25% of the amount of LKLF RNA that is produced in adult mouse lung ( Fig. 2A). Since LA-4 cells also exhibit many aspects of lung type II cells (Stoner et al., 1978) and type II cells express LKLF based on an in-situ analysis ( Wani, unpublished results),
Large deletions of the LKLF promoter sequence were created within the −2017 to +14 bp region, relative to the start of transcription. These deletion fragments were attached to a luciferase reporter gene in pGL3 (see Section 2). The constructs were then transfected into LA-4 and RAG cells, and the levels of luciferase expression were determined 24–48 h following transfection. Deletion of the region −2017/−1643 bp showed very little effect on luciferase reporter gene expression ( Fig. 2B). Further deletion to −490 bp increased expression in LA-4 cells to nearly double that of the −1643 bp construct. This suggested that the −1643/−490 bp region may contain negative cis-acting regulatory element(s). The deletion to −243 bp produces a fragment with the highest levels of expression when compared with all other deletion constructs. Finally, the expression from this promoter dropped to baseline levels when only 72 bp of 5∞ flanking sequence were included. These data indicate that major determinants of LKLF promoter specific transcription are located in the −243/−72 bp region when assayed in lung cell lines. Further characterization concentrated on this 172 bp region. The 172 bp fragment was subjected to a computerbased scan for consensus binding sites of characterized transcription factors (Collins and Driesel, 1991). Two Sp1 consensus binding sites were identified in this search. The first Sp1 binding site at −222 to −202 bp was designated Sp1A, and the second at −202 to −182 bp was designated as Sp1B (Fig. 3C ). Sp1 is a Kru¨ppel-like factor that binds to GC box promoter elements and is found in a wide variety of cells types ( Kadonaga et al., 1987). Although this factor is found ubiquitously, expression levels vary considerably, depending on the cell type (Pieler and Bellefroid, 1994). The position of the Sp1 binding site relative to other promoter elements has also been shown to affect tissue-specific gene expression (Gregory et al., 1996). Since these sites were the most prominent elements revealed by the computer analysis, we examined the Sp1 consensus binding sites first. 3.4. Nuclear binding assays and deletion analysis of the Sp1 sites. In order to determine whether the Sp1 consensus sites are capable of binding Sp1, an EMSA was performed using nuclear extract from LA-4 cells. Binding of crude
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Fig. 2. LKLF expression and deletion analysis in lung cell lines. (A) Northern blot containing 2 mg/lane Poly A+ RNA fractionated on 1% agarose gels was hybridized with a LKLF specific radiolabled probe. Expression was seen in LL/2, a mouse Lewis lung carcinoma and LA-4, a mouse lung adenoma cell lines. No expression was detected in H292, a human mucoepidermoid lung cell line. (B) LKLF expressions in lung tissue (intentionally underloaded), RAG and LA-4 cell lines were compared. RAG cells express LKLF at approximately 5% of LA-4 cell levels. Internal lanes from this Northern blot were deleted. A b-actin specific radiolabled probe was hybridized as a loading control in (A) and (B). (C ) LKLF promoter deletion constructs were used to identify cis- regulatory elements in LA-4 cells. Various promoter deletion constructs are shown on the left (see also Section 2). Relative levels of luciferase expression (as a percentage of the highest) are shown in a horizontal bar graph. The highest expressing construct −243 to +14 was arbitrarily placed at 100. The error bars represent the standard error and n>6 in all columns. RAG cells were used as negative LKLF expressing cell control for all constructs.
nuclear extract to the −243/−157 bp region produces several specific shifted fragments, as indicated by the arrows seen in Fig. 3. When unlabelled oligonucleotides corresponding to either Sp1A or Sp1B are added to the binding reaction mix, the complex is competed off. Nonspecific oligonucleotides have no effect. To determine whether the binding factor is indeed Sp1, a supershift assay with anti-Sp1 antibody was performed. Slower mobility complexes are evident in EMSAs with the presence of the Sp1 antibody indicating that Sp1 can
bind to at least one of the consensus sites Sp1A or Sp1B ( Fig. 3B). While Sp1 can bind to these sites, such findings do not address whether this binding is important in LKLF transcription. To determine whether these sites are involved in expression of the LKLF gene, these sites were removed and the resulting constructs assayed by transfection. Loss of the first Sp1 site by deletion to −202 bp reduces expression levels in LA-4 cells to 76.6% (SE+5.18) of −243 bp construct expression levels,
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Fig. 3. Upstream promoter characterization with LA-4 nuclear extract and Sp1 antibody. (A) The binding assay was performed using LA-4 nuclear extract and the −243 to −157 bp region of the promoter. The first and second lane represent free probe and probe bound to extract, respectively. Subsequent lanes are competed with 100× molar excess cold oligonucleotide. (B) Sp1 antibody supershift of the upstream region of the LKLF promoter. The first lane represents probe and LA-4 nuclear extract, and the second lane contains probe and rabbit pre-immune serum. The promoter fragment, LA-4 nuclear extract, and rabbit Sp1 Ab are shown in lane three. The arrow indicates the supershifted complex of the Sp1 nuclear factor. Note, this gel has been run longer to resolve the supershifted band. (C ) The sequence below is the LKLF promoter from −243 to +14. The TATA box and HindIII insertion site have been boxed to show the relative positions. The sequence corresponding to Sp1A is underlined in bold, and the sequence corresponding to Sp1B is double-underlined.
whereas the −157 bp deletion reduces expression levels in LA-4 cells to 65.8% (SE+4.88) of −243 bp levels. Thus, the Sp1 sites contribute modestly to expression levels but are not absolutely required for reporter expression. Moreover, a reduction in expression to base line levels is seen in RAG cells when the Sp1 sites are removed ( Fig. 2B, −243 construct).
3.5. Analysis of the −157 to −72 promoter region Since the Sp1 elements appear to play only a modest role, we further examined the region between −157/−72 bp for transcriptional elements. A TRANSFAC and TFSEARCH database analysis of this region did not reveal a strong consensus binding site
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Fig. 4. Four base pair mutagenesis of the LKLF promoter from −142 to −106 bp: a graphic representation showing transfected reporter constructs containing the LKLF promoter from −142 to −106. Regions M1, M2, and M3 are in bold and underlined and shown as wild-type ( W.T.) sequence. The mutations are in bold and underlined lower-case sequence beside the graph (A–I ). Error bars represent the standard error and n>6 in all columns. Black bars represent LA-4 cell, and gray bars represent RAG cell transfections.
that might confer lung gene expression. However, using the TFSEARCH website, two consensus binding sites in the −157/−72 bp fragment showed a sequence similarity with an element in the promoter for CDX1 and for TATA-binding proteins. Oligomers designed from the CDX1 sequence failed to compete the bound −157/−72 bp fragment from mouse lung nuclear extracts (data not shown). Moreover, hTAF p130 antibodies (for TATA-complex proteins) did not supershift lung extracts in similar EMSAs (data not shown). Finally, the human LKLF upstream sequence was compared with the murine sequence in Fig. 1 (data not shown, Wani et al., 1999). This identified a highly conserved sequence homology within the –157/−72 bp region. With no obvious candidate sequences, linker scanning mutagenesis was employed to search for potential functionally important elements. The 86 bp upstream region was mutated to produce a series of small replacement constructs within the −157 bp deletion. Mutational analysis of the −142/−106 bp region showed the greatest effect on expression in La-4 cells (data not shown). To further localize the cis-elements responsible for expression, regions M1, M2 and M3 were mutated in 4 bp increments using a 4 bp replacement strategy (Fig. 4). This analysis revealed separate regions responsible for reporter expression. The −138/−127 bp and
−122/−111 bp regions are critical, whereas mutation within the −126/−123 bp region has a more modest affect on gene expression ( Fig. 4). In addition, the TATA box of the LKLF promoter was identified by mutating an A to G and a T to C. In the context of the larger promoter (−243/+14), this mutation reduces the expression to 17.5% (SE+0.61) of the −243 reporter construct, indicating that the TATA box is required for LKLF transcription (data not shown). To determine whether nuclear factors bind to these functionally important regions, EMSAs were performed on the −157/−72 bp LKLF fragment using nuclear extracts from LA-4 and LL/2 cells. Three specific bands were observed (Fig. 5). When competed with either the A oligonucleotide or the E oligonucleotide, corresponding to the two sites critical for reporter expression (−138/−127 bp and −122/−111 bp), the two lower bands as well as a faint upper band disappear. Thus, specific competition with both the A and the E oligonucleotides can disrupt binding of nuclear factors to the −157/−72 bp region of the LKLF promoter. 3.6. Analysis of a core 30 bp promoter region Since the combined linker scanning and EMSA data suggest that an approximate 30 bp region is important for transcription, we investigated this region in more
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Fig. 5. Electrophoretic mobility shift assay of the −157/ −72 region of the LKLF promoter. Binding assays done on the −157/ −72 region of the LKLF promoter reveal a characteristic banding pattern (see arrows) that is disrupted when competed with 100× molar cold oligonucleotide A (−142 to −128 bp) or E (−125 to –108 bp). Binding with non-specific oligonucleotides shows no disruption in binding pattern (data not shown). Regions A and E correspond to the dark line and label in the sequence below the gel shift assay. Nuclear extract was prepared from LA-4 cells and LL/2 cells. A 5% non-denaturing polyacrylamide gel was run at 250 V for 2 h at 4°C
detail. We sought to determine whether the functional data from the LA-4 cell transfections would be reflected by a similar EMSA shift using adult lung tissue nuclear extracts. Under optimal binding conditions for this 30 bp fragment, a strong single specific band was seen with crude nuclear extract from adult lung ( Fig. 6A). This EMSA band showed a higher percentage of bound fragment (approximately 50%) than with the larger −157/−72 bp fragment and LA-4 nuclear extract (Fig. 5). A DNase I footprint analysis of this region with either the −157/−72 fragment or 30 bp region failed to show any binding residues. Furthermore, modifications using a methylation interference footprint on the 30 bp fragment also yielded no localization of a core binding sequence. To further determine the specificity of the protein complex binding to the 30 bp fragment, we mutated
oligonucleotides in three different regions of the −139/−110 bp binding sequence. Fig. 6A shows EMSAs containing oligonucleotides where the wild-type ( WT ) sequence is replaced with an EcoRI site (see Section 2.5). The M1 mutant oligonucleotide changes the 5∞ sequence (see Section 2.5) and causes a faster mobility complex shift of the protein–DNA interaction. The M2 and M3 mutant oligonucleotides change the middle 10 bases and the 3∞ sequence of the 30 bp fragment (see Section 2.5). No significant binding was seen with the M2 and M3 mutant oligonucleotides. These results suggest that the middle and 3∞ sequences are critical for overall binding and that the 5∞ region potentially binds a subset of proteins that bind to the wild-type fragment. Our linker-scanning cell expression functional data suggest that this 30 bp region is crucial for LKLF promoter expression in the LA-4 lung cell line. Moreover, the molecular binding of this 30 bp region is disrupted by site-specific mutagenesis. To determine whether the binding is specific for adult mouse nuclear extracts, we screened specific tissues that do and do not express LKLF using the wild-type 30 bp fragment. Fig. 6B shows EMSAs using adult brain, kidney, liver and lung nuclear extracts bound to the WT fragment. Extracts from liver and brain exhibit a similar mobility shift as seen with the lung extract. However, a lower mobility shift band is seen with kidney extract using the WT oligonucleotide. Interestingly, this lower shifted band is similar in size to the M1 mutant oligonucleotide ( Fig. 6A). Finally, to determine whether indeed a complex is formed in a differential manner, the WT and M1 oligouncleotides were bound with adult brain, kidney, liver and lung nuclear extract (Fig. 6B). All extracts with the M1 oligonucleotide appear to bind the same lower shifted band, as seen in kidney with the WT fragment. AP1 and SP1 oligonucleotide controls demonstrated that protein extracts were not degraded (data not shown). Collectively, this suggests that a protein(s) binding to the 5∞ most region of the 30 bp fragment has a differential expression pattern and appears to bind in a functionally important region.
4. Discussion The mouse LKLF gene is structurally related to the EKLF gene. There is conservation in the number of exons and the splice junctions. The 5∞ promoter regions vary considerably, however, as anticipated from the different expression patterns observed with these two transcription factors. In this study, it is shown that LKLF is expressed in lung tissue as well as in a number of cell lines derived from lung. In addition to this expression, we have observed comparable levels of LKLF expression in macrophage, endothelial and
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Fig. 6. (A) Electrophoretic mobility shift assay of the 30 bp (− 139/−110 bp) region of the LKLF promoter. Binding assays were done on the −139/−110 bp region of the LKLF promoter. A single shifted fragment (black arrow) was seen using the wild-type ( W ) sequence as labeled oligonucleotide and was competed away using cold fragment as before. The mutant M1 oligonucleotide shifted to a lower molecular weight fragment (gray arrow) than the W probe. Cold M1 marginally competes for the M1 probe, whereas W competes for nearly all the bound fragment. Mutant M2 and M3 probes failed to show significant binding. S, self-oligouncleotide; W, wild-type oligonucleotide. Adult lung nuclear extract was used. Bar schematic shows the position of the mutated oligonucleotides. (B) Tissue distribution EMSA of the 30 bp LKLF promoter fragment. Binding assays were done using a wild-type and mutant −139/−110 bp region of the LKLF promoter. Different tissues from nuclear extracts were tested with the wild-type ( W ) and mutant M1 probes. N, no extract; B, brain; K, kidney; Li, liver; Lu, lung. As in Fig. 6A, brain, liver and lung extract showed a similar band shift with the W probe (black arrow). However, a lower molecular fragment was seen with the kidney extract (gray arrow) using the W probe. All tissues tested showed the lower molecular weight shift with the M1 oligonucleotide probe (gray arrow). Competition oligonucleotides were as before (data not shown).
embryonic stem cell lines (data not shown). Our major interest is to describe the upstream regulators for LKLF expression and to identify specific cis-regulatory elements. The LA-4 murine lung cell line was chosen for analysis of LKLF promoter expression because it represents type II lung cells that express LKLF. Approximately 2 kb of the LKLF promoter were analyzed by deletion and linker scanning analysis. We observed one region with a repressive effect on reporter expression and one region of transcriptional activation. Although there is very little change between the −2017 and −1643 promoter deletion constructs, there is a twofold difference between the −1643 and −490 con-
structs. Within this 1153 bp segment are large regions of repeats and several transcription factor consensus binding sites. Interestingly, expression of the LKLF protein is able to overcome the negative regulation and restore the −2017 promoter construct to approximately −490 construct levels (data not shown). This activity could be derived from the binding of LKLF directly to the promoter and the activation of transcription in a manner consistent with other Kru¨ppel-like factors. Alternatively, LKLF could interact with negative transfactors to derepress expression. Precedence for such an interaction comes from studies demonstrating that some zinc finger proteins are able to associate with other
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transcription factors via the zinc finger domains (Crossley et al., 1995; Merika and Orkin, 1995; Gregory et al., 1996). The deletion of the putative Sp1 elements in the LKLF promoter results in a decrease in reporter gene expression. Sp1, also a Kru¨ppel-like factor that binds to GC box promoter elements ( Kadonaga et al., 1987), is found ubiquitously, but expression levels can vary 1000-fold, depending on the cell type (Pieler and Bellefroid, 1994). Tissue-specific gene expression can also be affected, depending on the Sp1 binding site position relative to other promoter elements (Gregory et al., 1996). In our study, deletion of the Sp1 elements reduces overall LKLF promoter activity but does not eliminate it. We conclude that Sp1 may be involved in the level of LKLF expression, but it is not critical for lung cell gene expression. Moreover, deletion of the Sp1 sites also reduces expression in RAG cells using the larger constructs. Therefore, the functional data indicate that these sites only contribute a modest activation as basal transcription elements. Mutations in the remaining sequence from −157/−72 produce different results when compared between LA-4 and RAG cells, indicating that this region is likely responsible for gene regulation. Linker scanning analysis of this region demonstrates that it is important for cell-line expression. Binding of LA-4 nuclear extracts confirms that proteins bind to this region. Attempts at obtaining a DNase I footprint in this region were unsuccessful, perhaps due to a low binding affinity of the larger fragment, the concentration of binding factor in the crude nuclear extract, or a labile protein complex. Mutational analysis, however, has shown that at least two regions are crucial for gene expression. Thus, when either one of the regions is mutated, the promoter is unable to direct cell-line expression of the reporter gene. The EMSA data correspond with the mutation studies in that competition with oligonucleotides derived from either of these regions abolishes specific binding to the entire −157/−72 probe. It is possible that one protein binds to the promoter region from −139/−110 and that −126/−123 are not essential for binding. This would also be consistent with the fact that the entire complex is disrupted when competed with either one or the other oligonucleotide. Another possibility is that two or more proteins bind to these regions, and binding of one requires binding of the other(s). The doublet pattern seen in gel shifts of longer run gels ( Fig. 5) could be indicative of more than one protein in the complex, or this could be the result of a modification such as phosphorylation of one or more of the binding proteins. Finally, using the 30 bp (−139/−110 bp) region, it is shown that mutated oligonucleotides cannot bind nuclear extract (Fig. 6A). The 5∞ region of this core element is not essential for binding as the remaining
wild-type 20 base sequence continues to bind protein(s). However, no protein binds to the mid- and 3∞ sequence when it is altered, suggesting that these regions are critical for binding of the protein complex overall. Base alterations of the 30 bp fragment, using DMS and DEPC footprinting methods, disrupted overall binding of extract to the probe. The base composition and sensitivity of this cis-regulatory region might also explain why our attempts to footprint the site failed. Critical nucleotides are likely altered so that no bound fragment can be obtained. Since several loosely conserved proteins that might bind to the −139/−110 region were ruled out, it is possible that a novel transcription factor binds to this site. In this study, a 2 kb promoter fragment 5∞ of the LKLF gene has been characterized. The upstream sequence includes a region from −157/−72 bp that is responsible for upregulating expression in LA-4 lung cell-line. Mutations within a 30 bp cis-element disrupt nuclear protein binding. Moreover, nuclear extracts from various tissues bind to this element in a differential manner. Although none of the cis-elements identified thus far exclusively binds to lung nuclear extracts, several functionally important regions do contribute to higher levels of reporter gene expression in the LA-4 lung cell lines. Identification of the factor(s) that bind the −139/−110 bp fragment awaits further isolation and characterization.
Acknowledgements This work was supported by NIH grants, HL57281 and POEMB HL41496. We thank Michael Conkright for the Northern blot data and helpful discussions from other members of the Lingrel laboratory.
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