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• W2079478580 abstract "In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5′-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in nonendothelial cells and interacts with endothelial and nonendothelial specific complexes that contain YY1. We demonstrate that β-actin is associated with YY1 specifically in the nucleus of nonendothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. In vivo analyses of the VWF promoter previously demonstrated that a fragment spanning sequences -487 to +247 targets promoter activation to brain vascular endothelial cells, whereas a longer fragment including 2182 bp of the 5′-flanking sequences, the first exon, and the first intron activated expression in endothelial cells of the heart and muscles as well as the brain of transgenic mice. These results suggested that additional VWF gene sequences were required for expression in other vascular endothelial cells in vivo. We have now identified a region within intron 51 of the VWF gene that is DNase I-hypersensitive (HSS) specifically in nonendothelial cells and interacts with endothelial and nonendothelial specific complexes that contain YY1. We demonstrate that β-actin is associated with YY1 specifically in the nucleus of nonendothelial cells and is a component of the nuclear protein complexes that interact with the DNase I-hypersensitive region. In vitro transfection analyses demonstrated that HSS sequences containing this YY1-binding site do not significantly affect VWF promoter activity. However, in vivo analyses demonstrated that addition of these sequences to the VWF promoter (-487 to +247) results in promoter activation in lung and brain vascular endothelial cells. These results demonstrate that the HSS sequences in intron 51 of the VWF gene contain cis-acting elements that are necessary for the VWF gene transcription in a subset of lung endothelial cells in vivo. von Willebrand factor (VWF) 2The abbreviations used are: VWFvon Willebrand factorHSSDNase I-hypersensitivekbkilobase pair(s)HUVEChuman umbilical vein endothelial cellHUMFIBhuman primary fibroblast(s)PBSphosphate-buffered salineI51-HSSintron 51-hypersensitive site(s)EMSAelectrophoretic mobility shift assayCCcommon complexHVCHUVEC complexHFCHUMFIB complex. 2The abbreviations used are: VWFvon Willebrand factorHSSDNase I-hypersensitivekbkilobase pair(s)HUVEChuman umbilical vein endothelial cellHUMFIBhuman primary fibroblast(s)PBSphosphate-buffered salineI51-HSSintron 51-hypersensitive site(s)EMSAelectrophoretic mobility shift assayCCcommon complexHVCHUVEC complexHFCHUMFIB complex. is an adhesive protein involved in healing wounds of the vasculature (1Wagner D.D. Annu. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar). The VWF gene, located on chromosome 12, is 178 kb long and contains 52 exons (1Wagner D.D. Annu. Rev. Cell Biol. 1990; 6: 217-246Crossref PubMed Google Scholar, 2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar). VWF is synthesized exclusively by endothelial cells and megakaryocytes. We have previously characterized a region of the VWF gene spanning sequences -487 to +247 that functions as an endothelial specific promoter in vitro. Transacting factors NF1, Oct 1, Ets, GATA6, HLP, NF-Y, and Ebp4 that positively and negatively regulate the activity of this promoter fragment were identified by others and us (3Hough C. Cuthbert C.D. Notley C. Brown C. Hegadorn C. Berber E. Lillicrap D. Blood. 2005; 105: 1531-1539Crossref PubMed Scopus (16) Google Scholar, 4Jahroudi N. Ardekani A.M. Greenberger J.S. J. Biol. Chem. 1996; 271: 21413-21421Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 5Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Crossref PubMed Scopus (107) Google Scholar, 6Peng Y. Jahroudi N. Blood. 2002; 99: 2408-2417Crossref PubMed Scopus (53) Google Scholar, 7Peng Y. Jahroudi N. J. Biol. Chem. 2003; 278: 8385-8394Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 8Schwachtgen J.L. Janel N. Barek L. Duterque-Coquillaud M. Ghysdael J. Meyer D. Kerbiriou-Nabias D. Oncogene. 1997; 15: 3091-3102Crossref PubMed Scopus (76) Google Scholar, 9Schwachtgen J.L. Remacle J.E. Janel N. Brys R. Huylebroeck D. Meyer D. Kerbiriou-Nabias D. Blood. 1998; 92: 1247-1258Crossref PubMed Google Scholar, 10Wang X. Peng Y. Ma Y. Jahroudi N. Blood. 2004; 104: 1725-1732Crossref PubMed Scopus (19) Google Scholar). However, in vivo analyses of this promoter region in transgenic mice demonstrated that it targets the expression of a fused LacZ gene to a subset of brain vascular endothelial cells in adult transgenic mice (11Aird W.C. Jahroudi N. Weiler-Guettler H. Rayburn H.B. Rosenberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4567-4571Crossref PubMed Scopus (100) Google Scholar). Additional sequences of the VWF gene that extended the 5′ region of the promoter to -2182 and the 3′ end of the promoter to the end of the first intron were shown to activate transcription in endothelial cells of the heart and muscles as well as the brain of transgenic mice (12Aird W.C. Edelberg J.M. Weiler-Guettler H. Simmons W.W. Smith T.W. Rosenberg R.D. J. Cell Biol. 1997; 138: 1117-1124Crossref PubMed Scopus (235) Google Scholar). These results suggest that distinct regions of the VWF gene are required to achieve promoter activity in the endothelial cells of distinct organs. von Willebrand factor DNase I-hypersensitive kilobase pair(s) human umbilical vein endothelial cell human primary fibroblast(s) phosphate-buffered saline intron 51-hypersensitive site(s) electrophoretic mobility shift assay common complex HUVEC complex HUMFIB complex. von Willebrand factor DNase I-hypersensitive kilobase pair(s) human umbilical vein endothelial cell human primary fibroblast(s) phosphate-buffered saline intron 51-hypersensitive site(s) electrophoretic mobility shift assay common complex HUVEC complex HUMFIB complex. To identify additional cis-acting elements within the VWF gene that may participate in transcriptional regulation, we explored the possibility that such sequences may be located in VWF chromatin regions that show hypersensitivity to DNase I. Many transcriptional regulatory elements are located within chromatin regions hypersensitive to DNase I and other nucleases. These hypersensitive regions usually contain binding sites for one or more transcription factors that can be either common or tissue-specific (13Boyes J. Felsenfeld G. EMBO J. 1996; 15: 2496-2507Crossref PubMed Scopus (145) Google Scholar). Using this approach we identified a region within intron 51 of the VWF gene that interacts with YY1-containing nuclear protein complexes and targets VWF promoter activation to a subset of lung vascular endothelial cells. Cell Culture and Transfection—Cell culture of HeLa and human umbilical vein endothelial cells (HUVECs) were carried out as previously described (5Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Crossref PubMed Scopus (107) Google Scholar). Human primary fibroblasts (HUMFIB) were maintained as described for HeLa cells. Sheep pulmonary artery endothelial cells were prepared as previously described (14Hoyt D.G. Mannix R.J. Rusnak J.M. Pitt B.R. Lazo J.S. Am. J. Physiol. 1995; 269: L171-L177PubMed Google Scholar) and maintained in Opti-MEM (Invitrogen) with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2. Transfections were performed using the Lipofectamine Plus system as described by the manufacturer (Invitrogen). Preparation of Nuclei, DNase I Digestion, and Southern Blot Analyses—Nuclei were prepared using a modification of a previously described procedure (15Richard-Foy H. Hager G.L. EMBO J. 1987; 6: 2321-2328Crossref PubMed Scopus (448) Google Scholar). Aliquots of nuclei containing 2 A260 units were treated with 0-15 units/ml RQ1 DNase I (Promega) for 10 min at 37 °C, and the reactions were quenched by addition of an equal volume of 25 mm EDTA, 2% SDS, and subjected to 200 μg/ml proteinase K digestion followed by RNase treatment. Isolated DNA was digested to completion with EcoRI and subjected to Southern blot analyses as described (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The probe for Southern blot analyses was synthesized using polymerase chain reaction with plasmid 2.3λ containing EcoRI fragment 38 of the VWF gene (2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar) (gift of J. E. Sadler) as template and the following primers: 5′-GGGACCTATTTCCAGCCCAGTGAG-3′ (forward) and 5′-CCCTCCGCACCCAGCCCTTATTG-3′ (reverse). Nuclear Extract Preparation, Electrophoretic Mobility Shift Analysis, Immunoprecipitation, and Western Blot Analyses—Nuclear extracts preparation, gel mobility, and supershift assays were carried out as previously described (4Jahroudi N. Ardekani A.M. Greenberger J.S. J. Biol. Chem. 1996; 271: 21413-21421Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 5Jahroudi N. Lynch D.C. Mol. Cell. Biol. 1994; 14: 999-1008Crossref PubMed Scopus (107) Google Scholar). Immunoprecipitation and Western blot analyses were performed as previously described (17Peng Y. Stewart D. Li W. Hawkins M. Kulak S. Ballermann B. Jahroudi N. Oncogene. 2007; 26: 7576-7583Crossref PubMed Scopus (26) Google Scholar). Antibodies used were mouse monoclonal anti-β-actin clone AC-15 (Sigma) and rabbit polyclonal anti-YY1 (Santa Cruz Biotechnology H-414). Mass spectrometry analyses were performed on the Coomassie Blue-stained polypeptide sliced from the gel using liquid chromatography-tandem mass spectrometry analyses by University of Alberta Peptide Institute as previously described (10Wang X. Peng Y. Ma Y. Jahroudi N. Blood. 2004; 104: 1725-1732Crossref PubMed Scopus (19) Google Scholar). Tandem mass spectrometry spectra were directly used to search the National Center for Biotechnology Information nonredundant protein data base with use of the mascot search program (Matrix Science, London, UK). Generation and Analyses of Transgenic Mice—DNA fragments containing HSS sequences, VWF-LacZ, and poly(A) were isolated and purified from the plasmids HSS-VWF-LacZ and VWF-LacZ-HSS (described in plasmids generation in supplementary material) by digestion with ASP718 (for plasmid HSS-VWF-LacZ) or SalI (for plasmid VWF-LacZ-HSS). Fragments were used for microinjection to generate C57BL/6 transgenic mice by Ozgene (Bentley, Australia) and University of Alberta transgenic mice facility, respectively. In addition DNA fragment containing VWF-LacZ transgene was used to generate transgenic mice containing VWF-LacZ gene in C57BL/6 mice by Ozgene. All animal housing and experimentation were approved by the Health Sciences Animal Policy and Welfare Committee at the University of Alberta. Two independent lines of HSS-VWF-LacZ and VWF-LacZ, as well as three independent lines of VWF-LacZ-HSS containing the transgenes (determined by PCR) were analyzed. These included one founder line of each HSS-VWF-LacZ and VWF-LacZ-HSS that did not produce progenies and F1 generation of the other lines. Organs, (lung, liver, kidney, brain, heart, stomach, duodenum, cecum, ear, and colon) of founders and F1 lines were harvested and fixed in 4% formaldehyde solution overnight followed by dehydration through graded alcohol, cleared, and tissue-banked as described (18Duta F. Ulanova M. Seidel D. Puttagunta L. Musat-Marcu S. Harrod K.S. Schreiber A.D. Steinhoff U. Befus A.D. Histochem. Cell Biol. 2006; 126: 495-505Crossref PubMed Scopus (20) Google Scholar). Tissue macroarrays were prepared by Histobest inc. (Edmonton, Canada). Sectioning was performed at 5 microns, and arrays were subjected to immunohistochemistry using primary anti-LacZ antibody (Abcam ab116-100) at a dilution of 1:500, and secondary anti-mouse IgG antibody (Dako Cytomation Carpinteria, CA) at a dilution of 1:2000 dilution. Normal Rabbit IgG antibody was used as negative control. The reaction products were detected with Envision+/horse-radish peroxidase (Dako Cytomation, Carpinteria, CA). Immunostained sections were counterstained with methyl green. In the mouse from one founder line of VWF-LacZ-HSS, the lungs were inflated with 2% paraformaldehyde and cryoprotected with 30% sucrose in phosphate-buffered saline (PBS). Subsequently, 5-micron cryosections were cut, permeabilized with Triton X-100 (0.2% v/v in PBS) for 10 min at room temperature, and washed three times with PBS. Tissue was incubated with rabbit anti-β-galactosidase (U. S. Biological) and rat anti-mouse cd31 (PECAM; clone 390; Pharmingen, San Diego, CA) antibodies for 1 h at room temperature, washed three times in PBS, and labeled with goat anti-rabbit Alexa 488 (Molecular Probes) along with goat anti-rat cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA). The slides were mounted in Gelvatol (Monsanto) and visualized using an Olympus 500 confocal microscope. The images were collected with a ×60 oil immersion objective at a 1,024 × 1,024 pixel resolution. The images were superimposed on differential interference contrast image for aid in structural resolution. β-Galactosidase and Luciferase Detection in Transfected Cells—Quantitative measurements of β-galactosidase and luciferase expression in transfected cells were performed using the β-Galactosidase Enzyme Assay System and Luciferase Assay System with Reporter Lysis Buffer (Promega) according to the manufacturer's directions. Immunofluorescence analysis was used to qualitatively detect β-galactosidase expression in transfected freshly isolated mouse pulmonary microvascular endothelial cells. For this analysis mouse pulmonary microvascular endothelial cells grown in Lab-Tek II chambered slides (Nunc) were fixed in 4% paraformaldehyde for 20 min and permeablized with 0.1%Triton X-100. The cells were stained with an Alexa 555-labeled rabbit anti-mouse polyclonal antibody to β-galactosidase (U. S. Biologicals) and Alexa 647-labeled IB4-isolectin (Molecular Probes). The images were collected using an Olympus Fluoview 1000 confocal microscope. Identification of a DNase I-hypersensitive Site within Intron 51 of the VWF Gene—Analysis of the VWF gene promoter and its 5′ proximal region in vivo demonstrated that additional regulatory DNA sequences are required for transcriptional activation of the VWF gene in endothelial cells of all organs. Although cis-acting regulatory elements may be present anywhere within the VWF gene, we chose to investigate whether the 3′ region of the VWF gene contained potential regulatory sequences. The rationale for choosing the 3′ region for this analysis was based on previous reports demonstrating that next to 5′ regions, regulatory elements are most commonly identified at the 3′ end of genes (19Mautner J. Joos S. Werner T. Eick D. Bornkamm G.W. Polack A. Nucleic Acids Res. 1995; 23: 72-80Crossref PubMed Scopus (32) Google Scholar, 20Radomska H.S. Satterthwaite A.B. Burn T.C. Oliff I.A. Huettner C.S. Tenen D.G. Gene (Amst.). 1998; 222: 305-318Crossref PubMed Scopus (24) Google Scholar, 21Roque M.C. Smith P.A. Blasquez V.C. Mol. Cell. Biol. 1996; 16: 3138-3155Crossref PubMed Google Scholar). We employed the technique of DNase I-hypersensitive site identification commonly used to locate potential DNA regulatory sequences within large genomic regions. Nuclei were prepared from HUVECs that express VWF and two nonendothelial cell types, HeLa and HUMFIB, that do not express VWF. Isolated nuclei were either untreated or subjected to increasing concentrations of DNase I prior to digestion with EcoRI restriction enzyme and DNA isolation. DNA was then subjected to Southern blot analysis using a radioactively labeled 233-bp DNA probe corresponding to nucleotides 38/3 to 38/235 of the VWF gene (2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar), including regions of intron 50, exon, and intron 51 (Fig. 1A). Based on the previously reported sequence of the VWF gene (2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar), we expected to detect a 2.4-kb EcoRI DNA fragment following hybridization of the sample that was not treated with DNase I. In both the HUVEC and HUMFIB samples, a 2.4-kb fragment was observed (Fig. 1B, 2.4 kb PB). However, in HUMFIB DNA, an additional fragment of ∼300 bp was also detected (Fig. 1B, 0.3 kb DNB). The 300-bp fragment was observed even without addition of DNase I, suggesting that this site is sensitive to endogenous nucleases. Similar results were obtained when HeLa chromatin was analyzed (data not shown). For HUVEC, although increasing the concentration of DNase I treatment resulted in a decrease in intensity of the 2.4-kb fragment, the level of decrease was significantly lower than that for HUMFIB nuclear DNA, and the 300-bp fragment was not detected (Fig. 1B, lanes 5-9). These results demonstrated the presence of a strong, DNase I-hypersensitive site within intron 51 of the VWF gene in two human, nonendothelial cell types (HeLa and fibroblasts) that was absent in HUVEC. Identification of Specific Protein-DNA Binding Sequences within the 3′ Fragment—Based on detection of the VWF intron 51-hypersensitive site in nonendothelial cells and its absence in endothelial cells, we hypothesized that the DNase 1 hypersensitivity may have arisen as a result of DNA-nuclear protein interactions that may function as repressors in nonendothelial cells. To test this hypothesis, we first determined whether specific protein-DNA complex formation occurs at the intron 51-hypersensitive site (I51-HSS) in nonendothelial cells. We carried out electrophoretic mobility shift assays (EMSA) utilizing nuclear extracts from human fibroblast cells and a 205-bp probe encompassing the DNase I-hypersensitive site. The probe corresponded to sequences 38/231 to 38/435 of the VWF gene (2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar). Several protein-DNA complexes (Fig. 2, C1-C3) formed with this fragment that were abolished or significantly reduced in the presence of specific competitor, whereas a nonspecific DNA competitor did not effect these complex formations (Fig. 2, lanes 1-3). To localize the binding sequences of the nuclear protein(s) in this 205-bp DNA sequence, smaller DNA fragments corresponding to various regions within this DNA fragment were used as competitors in EMSA. Specific protein-DNA interactions sites (specifically complexes C2 and C3) were localized to nucleotides 38/380 to 38/409 (Fig. 2, lane 9), and another protein-DNA interaction site (complex C1) appeared to be present in nucleotides 38/281 to 38/380 (Fig. 2, lane 5). The complex C1 was not consistently observed in repeated assays. Thus based on consistency and the prominence of the specific protein-DNA interactions that occurs with nucleotides 380-409, we first chose to pursue characterization of this protein complex. Protein Complexes That Include YY1 Transcription Factor Interact with DNA Sequences in the Intron 51 DNase I-hypersensitive Region—Based on observations that I51-HSS was specifically detected in nonendothelial cells and the hypothesis that the proteins interacting with I51-HSS sequences may function as a repressor, we proceeded to determine whether there are differences in the presence/absence or the pattern of the observed specific protein-DNA complexes that formed with nucleotides 380-409 in nonendothelial compared with endothelial cells. We carried out gel mobility experiments using a double-stranded oligonucleotide corresponding to nucleotides 38/380-38/409 as a probe and nuclear extracts from HUVECs and HUMFIB and HeLa cells. The results demonstrated that nuclear proteins from both cell types form a complex that migrates to a similar position in an acrylamide gel (referred to as common complex (CC)). In addition to this, cell type-specific complexes were formed with HUVEC and HUMFIB nuclear extracts (referred to as HUVEC complex (HVC) and HUMFIB complex (HFC), respectively (Fig. 3A)). HeLa cells nuclear extracts also demonstrated a pattern of complex formation similar to that observed for HUMFIB cells (Fig. 3A, lane 8). To identify the nucleotides within the 380-409 DNA fragment necessary for the formation of various complexes, we generated additional sets of oligonucleotides containing different four nucleotide substitutions (Fig. 3C). The only mutant oligonucleotide that did not significantly compete with the wild type probe and hence was unable to abolish complex formations was the m2 oligonucleotide (Fig. 3B). Based on these results, a core DNA sequence, AATG, was identified as necessary for formation of specific protein-DNA complexes. These results also demonstrated that the same core sequence was necessary for the formation of all three (CC, HVC, and HFC) complexes. Using a data base containing transcription factor binding consensus sites, we determined that the sequence AATGG and the surrounding nucleotides (AA AATG G) were homologous to the core consensus binding site of the transcription factor YY1 (Yin Yang 1). To determine whether YY1 was interacting with this core sequence, we carried out competition and supershift experiments using sequences 380-409 as probe, oligonucleotides containing the consensus YY1 binding sequence (corresponding to that of murine immunoglobulin heavy-chain intronic enhancer (μE1)) as competitor, and anti-YY1 antibody. The results demonstrated that all three complexes were abolished in the presence of the YY1-specific competitor (Fig. 4A, lanes 3 and 5) and were supershifted in presence of the anti-YY1 antibody (Fig. 4B). These data demonstrate that YY1 interacts with the intron 51 sequence AATGG, and both the common and cell specific complexes formed with HUVEC and human fibroblast nuclear extracts contain YY1. Intron 51 HSS Sequences Target the Activation of the VWF Promoter in a Subset of Lung Endothelial Cells in Vivo—VWF promoter sequences -487 to +247 activate transcription only in endothelial cells of the brain but not other organs. To determine whether the 380-bp DNA region (referred to as I51-HSS) that encompasses the DNase I-hypersensitive and YY1-binding site participates in regulation of VWF transcription in vivo, we generated transgenic mice harboring a LacZ transgene driven by the VWF promoter (sequences -487 to +247) and the I51-HSS sequences (spanning nucleotides 38/231 to 38/613 (2Mancuso D.J. Tuley E.A. Westfield L.A. Worrall N.K. Shelton-Inloes B.B. Sorace J.M. Alevy Y.G. Sadler J.E. J. Biol. Chem. 1989; 264: 19514-19527Abstract Full Text PDF PubMed Google Scholar)) positioned either upstream of the VWF promoter or downstream of the LacZ gene (transgenes HSS-VWF-LacZ and VWF-LacZ-HSS; Fig. 5A). We analyzed two and three independent lines of each transgenic mouse, respectively, using immunohistochemistry and immunofluorescence to detect LacZ gene products in various organs. We analyzed one founder line and F1 generation of other lines, for each transgene. Organs from transgenic and nontransgenic litter mates were harvested and fixed in paraformaldehyde or cryopreserved in 30% sucrose (one founder line for VWF-LacZ-HSS). Tissue arrays were prepared from paraformaldehyde-treated organs and subjected to immunohistochemistry with anti-β-galactosidase-specific antibody. The use of tissue arrays in which sections of various organs were placed on a single slide allows for samples to be simultaneously processed under a similar condition, thus minimizing interexperimental staining variability. Positive immunoperoxidase staining for β-galactosidase was shown almost exclusively in the vasculatures of lung and brain (Figs. 5 and 6) for both HSS-VWF-LacZ and VWF-LacZ-HSS. In the lung, vessels of the alveolar walls, especially in the parenchyma near the pleura, were most commonly positive. Specific staining of the endothelial cells of lung and brain tissues of the transgenic lines with control IgG antibody and nontransgenic littermates with the anti-β-galactosidase antibody was not observed (Fig. 6, D and E). Also consistent with our previous report, there was no LacZ detection in the lung of transgenic mice (VWF-LacZ) that contained VWF promoter (-487 to +247) in the absence of the I-51HSS sequences, whereas expression was observed in the brain vasculature of these animals (Fig. 6C).FIGURE 6Hypersensitive region of the VWF gene intron 51 when placed downstream of the VWF promoter also targets the promoter activity to the lung vasculature in transgenic mice. Sections from lung and brain of the transgenic mice (F1 generations) HSS-VWF-LacZ (A), VWF-LacZ-HSS (B), VWF-LacZ (C), and a nontransgenic littermate (E) were immunostained with an antibody specific to LacZ. The lung and brain sections of HSS-VWF-LacZ transgenic mouse were also immunostained with an isotype-matched IgG antibody used as negative control (D). The arrows show representative vessels expressing LacZ (magnification, 600×). F, confocal microscopy analysis of LacZ (4Jahroudi N. Ardekani A.M. Greenberger J.S. J. Biol. Chem. 1996; 271: 21413-21421Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and PECAM (red) showing overlapping LacZ and PCAM expression in brain and lung endothelial cells of a VWF-LacZ-HSS transgenic mouse line 1132. Similar to other lines, expression was not detected in vasculature of other organs in this line as well (data not shown). WT, wild type; β-gal, β-galactosidase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine the cellular origin of the LacZ expression in the lung vasculature, immunofluorescent analysis was performed on OCT frozen organs harvested from one line of VWF-LacZ-HSS using specific antibodies to detect LacZ gene product (shown as green) and PECAM (shown as red, a marker for endothelial cells), and slides were subjected to confocal fluorescence microscopy. Overlapping windows (yellow) demonstrated colocalization of both PECAM and β-galactosidase in brain and lung endothelial cells (Fig. 6F). Similarly LacZ expression was not detected in endothelial cells of other organs tested (data not shown). Some β-galactosidase staining was observed in the colon (nonendothelial cells) of one line of HSS-VWF-LacZ and the kidney (nonendothelial cells) of one line of VWF-LacZ-HSS. These were attributed to ectopic expression, possibly caused by transgene integration site, because these were not observed in more than one transgenic line each. There was no other detection of β-galactosidase in endothelial or nonendothelial cells of other organs tested. Also there was no expression detected in the megakaryocytes (data not shown). The results demonstrated that LacZ transgene was expressed only in the brain and lung vasculatures of these mice and that the expression was restricted to endothelial cells of these organs. Because the VWF promoter sequence -487 to +247 was previously shown to target activation in brain vascular endothelial cells, these results demonstrate that the lung-endothelial specific activation of the chimeric promoter, containing I51-HSS sequences and sequences -487 to +247, is due to the regulatory function of the I51-HSS sequences. The Effect of the Intron 51 HSS Sequences on Gene Expression Driven from Homologous and Heterologous Promoters in Endothelial and Nonendothelial Cells in Vitro—To determine whether I51-HSS sequences have a differential and potentially cell type-specific function in regulating VWF promoter in endothelial and nonendothelial cell types, we determined the transcriptional activity of the homologous VWF promoter (sequences -487 to +247) and a heterologous SV40 early promoter in the absence and presence of I51-HSS sequences, in HUVEC and sheep pulmonary endothelial cells as well as HeLa cells. Because VWF promoter fragment (-487 to +247) is not activated in nonendothelial cells, to test the hypothesis that I51-HSS may function as a repressor in nonendothelial cells, we chose to determine its effect on the activity of a heterologous SV40 promoter. Plasmids containing human gro" @default.
• W2079478580 created "2016-06-24" @default.
• W2079478580 creator A5036211829 @default.
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• W2079478580 date "2008-02-01" @default.
• W2079478580 modified "2023-10-16" @default.
• W2079478580 title "Sequences in Intron 51 of the von Willebrand Factor Gene Target Promoter Activation to a Subset of Lung Endothelial Cells in Transgenic Mice" @default.
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