Promoter Regulation of the Visinin-like Subfamily of Neuronal Calcium Sensor Proteins by Nuclear Respiratory Factor-1
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.049361
ISSN1083-351X
AutoresJian Fu, Jirong Zhang, Fang Jin, Jamie Patchefsky, Karl‐Heinz Braunewell, Andres J. Klein–Szanto,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoVILIP-1 (gene name VSNL1), a member of the neuronal Ca2+ sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5′ terminus disclosed that the last 3′ terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5′ terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/α-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD. VILIP-1 (gene name VSNL1), a member of the neuronal Ca2+ sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5′ terminus disclosed that the last 3′ terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5′ terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/α-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD. VILIP-1 3The abbreviations used are: VILIP-1visinin-like protein-1HDAChistone deacetylaseEWGerect wing geneNCSneuronal Ca2+ sensorChIPchromatin immunoprecipitation, DN dominant-negativeNRF-1nuclear respiratory factor-1EMSAelectrophoretic mobility shift assayVPVSNL1 promotersiRNAsmall interfering RNAMBDmethyl-CPG-binding domainMBPmethyl-binding proteins. (visinin-like protein-1, gene name VSNL1) is a member of the visinin-like subfamily of neuronal Ca2+ sensor (NCS) proteins, which include neurocalcin-δ (NCALD), hippocalcin (HPCA), VILIP-1, VILIP-2 (HPCAL4), and VILIP-3 (HPCAL1) (1Braunewell K.H. Trends Pharmacol. Sci. 2005; 26: 345-351Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 2Burgoyne R.D. Nat. Rev. 2007; 8: 182-193Crossref Scopus (418) Google Scholar, 3Braunewell K.H. Klein-Szanto A.J. Cell Tissue Res. 2009; 335: 301-316Crossref PubMed Scopus (89) Google Scholar). In the nervous system, VILIP-1 was consistently found to be expressed in hippocampal neurons, cerebellar granular cells, interneurons, cortical pyramidal cells, as well as in other neurons (4Braunewell K.H. Gundelfinger E.D. Cell Tissue Res. 1999; 295: 1-12Crossref PubMed Scopus (236) Google Scholar, 5Bernstein H.G. Baumann B. Danos P. Diekmann S. Bogerts B. Gundelfinger E.D. Braunewell K.H. J. Neurocytol. 1999; 28: 655-662Crossref PubMed Scopus (102) Google Scholar). The deregulation of VILIP-1 expression implicates it in certain pathological processes of the nervous system such as Alzheimer disease and schizophrenia (1Braunewell K.H. Trends Pharmacol. Sci. 2005; 26: 345-351Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). visinin-like protein-1 histone deacetylase erect wing gene neuronal Ca2+ sensor chromatin immunoprecipitation, DN dominant-negative nuclear respiratory factor-1 electrophoretic mobility shift assay VSNL1 promoter small interfering RNA methyl-CPG-binding domain methyl-binding proteins. Although VILIP-1 was originally identified in the brain, its expression can be detected in the peripheral tissues and organs such as heart, lung, liver, and testis (6Gierke P. Zhao C. Brackmann M. Linke B. Heinemann U. Braunewell K.H. Biochem. Biophys. Res. Commun. 2004; 323: 38-43Crossref PubMed Scopus (34) Google Scholar), suggesting a potential role in maintaining normal tissue homeostasis in both nervous tissue and non-neural tissues. Recently, VILIP-1 was found to be expressed in insulin-expressing pancreatic β-cells and glucagon-expressing α-cells (7Dai F.F. Zhang Y. Kang Y. Wang Q. Gaisano H.Y. Braunewell K.H. Chan C.B. Wheeler M.B. J. Biol. Chem. 2006; 281: 21942-21953Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Basal epidermal keratinocytes of normal murine skin express VILIP-1, whereas its expression is markedly decreased in aggressive and invasive squamous cell carcinomas (8Mahloogi H. González-Guerrico A.M. Lopez De Cicco R. Bassi D.E. Goodrow T. Braunewell K.H. Klein-Szanto A.J. Cancer Res. 2003; 63: 4997-5004PubMed Google Scholar). Enforced expression of VILIP-1 led to inhibition of cell adhesion and migration by down-regulating fibronectin receptors, suggesting a tumor suppressor function for VILIP-1 (9Gonzalez Guerrico A.M. Jaffer Z.M. Page R.E. Braunewell K.H. Chernoff J. Klein-Szanto A.J. Oncogene. 2005; 24: 2307-2316Crossref PubMed Scopus (29) Google Scholar). Similarly, VILIP-1 expression is present in normal squamous epithelium of esophagus and bronchial mucociliary epithelium. Conversely VILIP-1 was down-regulated in squamous cell carcinomas from these two sites (10Wickborn C. Klein-Szanto A.J. Schlag P.M. Braunewell K.H. Mol. Carcinog. 2006; 45: 572-581Crossref PubMed Scopus (15) Google Scholar, 11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). Despite the recent reports that highlight the role of VILIP-1 in physiological and pathological processes, the molecular mechanisms controlling its expression remain unknown. Recently, we reported the cloning of a 2-kb VSNL1 promoter from the 5′-untranslated region of the gene (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). The VSNL1 2-kb promoter contains two CpG islands that are the targets of methylation modification, constituting one of the epigenetic mechanisms contributing to the silencing of VSNL1 gene expression in lung and other cancer cells. Increased acetylation of histones 3 and 4 around the VSNL1 promoter by the histone deacetylase inhibitor, trichostatin A, released the inhibition of gene expression, thus leading to the reactivation of VSNL1 expression (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). To further understand VSNL1 gene regulation and identify the transcriptional elements of the human VSNL1 promoter, we characterized the 2-kb VSNL1 promoter by deletion, mutation, and in vitro DNA binding and chromatin immunoprecipitation assays. Using dominant-negative construct transfection and siRNA knockdown techniques, we identified Nuclear respiratory factor 1 (NRF-1) as a major trans-acting element regulating VSNL1 promoter. In addition, we found that NRF-1 could be the regulatory factor for the promoters of all the other visinin-like subfamily genes. Two non-small cell lung cancer cell lines, NCI-H522 and NCI-H520, which express different levels of VILIP-1, and normal human bronchial epithelial cells were cultured as previously described (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). pGL4.10[luc2] vector and pGL4.73 luciferase reporter vectors (Promega, Madison, WI) were used for VSNL1 promoter reporter assays. VP-1998 (VP2kb) was constructed as described (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). All of the deletion constructs of VSNL1 promoter were derived from VP-1998 by PCR using the same reverse primer: 5′-GAAGATCTGCAGATTGGGAATCCCATAG. The forward primers were as follows: for VP-354, 5′-GGGGTACCAGGTTCGGTAACCACTGGG; for VP-174, 5′-GGGGTACCTGCGCCATCGCCAGGCG; for VP-100, 5′-GGGGTACCAAGAGAGGAAAGGGGAGGG; for VP-90 5′-GGGGTACCAGGGGAGGGGGTGCCTG; for vp-80, 5′-GGGGTACCGTGCCTGGAGAGGCGGAG; for VP-70, 5′-GGGGTACCAGGCGGAGGCTCGCGCG; for VP-60, 5′-GGGGTACCTCGCGCGCCTGCGCATC; and for VP-50, 5′-GGGGTACCGCGCATCCAGCTCCAGGG. For VP-100 and VP-90 containing Sp1 mutations and VP-60 containing NRF-1 mutation, the same reverse primer as above was used. The forward primers were: for VP-100Sp1m, 5′-GGGGTACCAATTTAGGAAAGGGGAGGGGGTG; for VP-90Sp1m, 5′-GGGGTACCAGTTTAGGGGGTGCCTGGAGAGG; and for VP-60NRFm, 5′-GGGGTACCTCGCGCGGCATTCCATCCAGCTCC. The PCR products were digested with KpnI and BglII and then inserted into pGL4.10 basic vector. VP-174NRFm, in which the NRF-1-binding site was mutated, was made by using QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) with the following primers: forward, 5′-GGCGGAGGCTCGCGCGGCATTCCATCCAGCTCCAGGG; and reverse, 5′-CCCTGGAGCTGGATGGAATGCCGCGCGAGCCTCCGCC. The underlined bases were mutated within NRF-1 sites. All of the constructs had been verified by DNA sequencing. The expression plasmid of DN NRF-1 was constructed according to Chang and Huang (12Chang W.T. Huang A.M. J. Biol. Chem. 2004; 279: 14542-14550Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The DN NRF-1 consisted of the first N-terminal 304 residues of NRF-1, which contained the DNA-binding and nuclear localization domains and lacked the transactivation domain. DN NRF-1 cDNA was amplified from RNA extracted from normal human bronchial epithelial cells by using the SuperScript One-Step reverse transcription-PCR system (Invitrogen). The following primers were used: 5′-TTAAGCTTGCGCAGCCGCTCTGAGAAC and 5′-GACTCGAGTCACTGTGATGGTACAAGATGAGC. The underlined regions indicate the restriction enzyme sites. The cDNA fragments were digested with HindIII and XhoI and inserted into pcDNA3.1B+Myc vector (Invitrogen) to make pCDNA3.1 Myc-DN-NRF-1. The transfection of this construct resulted in the expression of Myc-tagged DN NRF-1 (therefore named Myc-DN-NRF-1). For all experiments, the cells were transfected by Lipofectamine 2000 (Invitrogen) using the manufacturer's protocol. For reporter gene assay, DNA mixture containing 0.8 μg of VSNL1 promoter constructs and 8 ng of pGL4.73, a transfection efficiency control, was diluted in 50 μl of Opti-Mem I medium (Invitrogen) and mixed with 2 μl of Lipofectamine 2000 diluted in 50 μl of Opti-Mem I medium. 100 μl of DNA-Lipofectamine 2000 complexes were added to each well of 24-well plates after 20 min of incubation at room temperature, and the transfected cells were left in the incubator for 24 h before the lysis step with 150 μl of passive lysis buffer. Reporter gene activity was measured in 96-well plates according to the protocol from Dual-Luciferase reporter 1000 assay system kit (Promega) by using an Envision plate reader (PerkinElmer Life Sciences). For assessing the effect of DN NRF-1 on VSNL1 promoter activity, different amounts of pCDNA3.1 Myc-DN-NRF-1 or vector were co-transfected with 0.8 μg of pGL4.10 VP-174 and 8 ng of pGL4.73, and luciferase activity was measured in 1 or 2 days. For siRNA transfections, NRF-1 siRNA (SC-38105) and scrambled control siRNA (SC-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Different amounts of NRF-1 siRNA were tested for knocking down NRF-1 protein, and 50 pmol was finally used in the reporter gene assay with 0.8 μg of VP-174 and 8 ng of pGL4.73 in 24-well plate. Luciferase activity was measured in 3 days. For immunoblot analysis of VILIP-1 protein after NRF-1 knockdown, scrambled (D-001810-10-05) and NRF-1 (L-017924-00-0005) siRNAs from Thermo SCIENTIFIC were also used. All of the data were obtained from at least three individual experiments. Nuclear proteins were extracted from cells using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) according to the protocol provided by the company. The following NRF-1 oligonucleotides were used: forward, 5′-TCGCGCGCCTGCGCATCCAG, and reverse, 5′-GCTGGATGCGCAGGCGCGCG; for methylated NRF-1 M1: forward, 5′ TCGCMetGCMetGCCTGCMetGCATCCAG, and reverse, 5′-GCTGGATGCMetGCAGGCMetGCMetGCG; and for methylated NRF-1 M2: forward, 5′-TCGCGCMetGCCTGCMetGCATCCAG, and reverse, 5′-GCTGGATGCMetGCAGGCMetGCGCG. CMet represents the methylated cytosine. The equal volumes of the forward and reverse oligonucleotides with the same molar concentration were mixed and boiled for 10 min and were annealed at room temperature overnight. The probe labeling and EMSA were performed using a gel shift assay system (Promega). Briefly, the annealed double-stranded oligonucleotides were labeled using T4 polynucleotide kinase before purification with Microspin G-25 columns (GE Healthcare, Buckinghamshire, UK). The DNA binding was conducted at 4 °C for 30 min in a mixture containing 5 μg of nuclear extract, 1× gel shift binding buffer, and 1 μl of 32P-labeled probe. In the competition experiment, 60× unlabeled double-stranded oligonucleotides were used as competitors and incubated with nuclear extract and binding buffer for 30 min before the addition of 32P-labeled probe. The reactions were further incubated at 4 °C for 30 min. In supershift assays, the antibodies were also incubated with the reaction mixture at 4 °C for 30 min before the addition of the 32P-labeled probe. Anti-NRF-1 antibodies were kindly provided by Dr. Claude Piantadosi (13Piantadosi C.A. Suliman H.B. J. Biol. Chem. 2008; 283: 10967-10977Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) and Dr. Kimitoshi Kohno (14Izumi H. Ohta R. Nagatani G. Ise T. Nakayama Y. Nomoto M. Kohno K. Biochem. J. 2003; 373: 713-722Crossref PubMed Google Scholar). The control sc2025 mouse IgG was from Santa Cruz Biotechnology. Finally the reaction mixtures were analyzed on a 6% DNA retardation gel (Invitrogen) at 10 v/cm on ice for 1.5 h. The gel was dried and analyzed by autoradiography. ChIP was performed as before (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar) using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Lake Placid, NY) following the manufacturer's protocol. Briefly, the proteins were cross-linked to DNA by incubating 1 × 106 cells with 1% formaldehyde for 2.5 or 10 min at 37 °C. The cell pellets were resuspended in 200 μl of SDS lysis buffer followed by DNA sonication for a total of 16 times (each time for 20 s at 30% of maximal power) by using the Ultrasonic Processor (Cole Parmer, Vernon Hills, IL). Immunocomplexes were captured from the rest of the lysates with 10 μg of antibody. After the cross-linking was reversed by heating the sample at 65 °C for 4 h, DNA was then extracted with phenol/chloroform and precipitated with ethanol. PCR was performed by using 1% of immunoprecipitated material and the same primers as used before (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). The following PCR program was used: 95 °C for 5 min followed by 35–40 cycles of 95 °C for 35 s, 54 °C for 45 s, 72 °C for 40 s, and finally 72 °C for 10 min. Rabbit anti-NRF-1 serum was kindly provided by D. Reines (15Smith K.T. Coffee B. Reines D. Hum. Mol. Genet. 2004; 13: 1611-1621Crossref PubMed Scopus (30) Google Scholar). Antibodies from Santa Cruz Biotechnology include: normal rabbit IgG (sc-2027), MBD4 (sc-10753), HDAC1 (sc-7872 X), HDAC2 (sc-7899 X), and HDAC3 (sc-11417 X). Antibodies against four MBP family proteins were from Abcam, including MBD1 (ab3753), MBD2a (ab3754), MBD3 (ab3755), and MeCP2 (ab3752). Anti-human α1-antitrypsin rabbit serum (A0409) was from Sigma. The promoter-specific methylation of VSNL1 was performed according to a published protocol (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). In this case we used HhaI DNA methyltransferase (New England Biolabs, Ipswich, MA) in the methylation-modifying treatment. VP-100 was used because it contains the NRF-1 site, which is the only target site within VP-100 for HhaI DNA methyltransferase. The HhaI methylation of the whole plasmids pGL4.10 and pGL4.10VP-100 was done as follows: 20 μg of plasmids was treated with HhaI DNA methyltransferase in the presence or absence of S-adenosylmethionine and gel-purified by using a QIAquick gel extraction kit (Qiagen). The complete methylation of VP-100 or plasmids was confirmed by digestion with HhaI restriction enzymes (New England Biolabs). The modified plasmids were co-transfected with pGL4.73 as described above. Immunoblotting analysis of protein was performed as described (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). The expression of DN NRF-1 was detected by using 25 μg of nuclear proteins and anti-Myc antibody (R950-25; Invitrogen). NRF-1 anti-serum (15Smith K.T. Coffee B. Reines D. Hum. Mol. Genet. 2004; 13: 1611-1621Crossref PubMed Scopus (30) Google Scholar) was kindly provided by Dr. D. Reines (Emory University, Atlanta, GA) and used to detect NRF-1 protein. The fragments containing the potential NRF-1-binding site were amplified by PCR using the following primers. For NCALD, the forward primer is 5′-GGGGTACCACCATCTGGAATGGGAGTTG; and the reverse primer is 5′-GAAGATCTAGTATGCTGTGGCACAAACG. For HPCA, the forward primer is 5′-GGGGTACCTGAGGGCGTCCCCTTCTC; and the reverse primer is 5′-GAAGATCTGACTGCGCAGGGAAGGCG. For HPCAL4, the forward primer is 5′-GGGGTACCGCAGAGATGTGGACTGCTG; and the reverse primer is 5′-GAAGATCTCCGGTGGGTTTGTTGC. For HPCAL2, the forward primer is 5′-GGGGTACCCTGGCCCCTCCCGGGC; and the reverse primer is 5′- GAAGATCTGCAAAGAGCCGGATCGCAG. The fragments were subsequently subcloned to pGL4.10[luc2] vector. Transcription factor binding sites were searched by using the following programs: TESS (Current Protocols in Bioinformatics), AliBaBa2.1 (Biostatics and Bioinformatics Facility, Fox Chase Cancer Center), and MatInspector (Genomatix). For comparing the relative activity of different reporter constructs, unpaired Student's t test for paired comparisons was performed, and a p value <0.05 was considered as significant. In our previous study, we had cloned a 2-kb fragment of VSNL1 promoter (VP-1998) from normal human bronchial epithelial cells and showed that its regulation by epigenetic mechanisms included promoter hypermethylation and histone acetylation (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). To define the boundaries of the minimal promoter and to identify cis elements that govern the transcriptional activity of VSNL1, we prepared a series of truncation constructs (Fig. 1A) and tested them in a transient luciferase reporter system. As shown in Fig. 1 (B and C), VP-354 was observed to have stronger transcriptional activity than VP-1998 in two non-small cell lung cancer cell lines (NCI-H522 and NCI-H520), indicating that putative negative cis-acting regulatory elements are located between the −1998 and −354. A 5′-deletion of 180 bp (VP-174) reduced promoter activity to the level comparable with that of VP-1998 in NCI-H522 cells. However, in the other non-small cell lung cancer cell line (NCI-H520), the reduction of activity was not as remarkable as in NCI-H522 cells. Further deletion of 74 bp from VP-174 caused a 14% decrease in activity when using NCI-H522 cells. In contrast, a 37% increase in activity was noted with NCI-H520 cells. VP-50 containing only the last 50 bp displayed the lowest luciferase activity in both cell lines. Its activity was 4 and 35% of VP-1998 in NCI-H522 and NCI-H520 cells, respectively, strongly indicating that the region from VP-100 to VP-50 is critical to VSNL1 promoter activity. To further examine which part within the truncated 50 bp could sustain transcriptional activity, we designed 10-bp stepwise deletion constructs (Fig. 1D) and tested them in the same assay system. The stepwise 10-bp deletion caused a gradual loss of promoter activity, and VP-60 still maintained 26 and 43% of VP-100 activity in NCI-H522 and NCI-H520, respectively (Fig. 1, E and F). Nevertheless, removal of 10 bp from VP-60 dramatically decreased the activity to 6 and 14% of VP-100 in NCI-H522 and NCI-H520 cells, respectively. We searched the sequence of VP-100 for homology to known regulatory elements by using the TESS, AliBaBa2.1, and MatInspector bioinformatics programs. There were a number of putative binding sites for transcriptional factors in this region (Fig. 2), among which Sp1 and NRF-1 were frequently identified. Combining this in silico analysis with the result from the 10-bp stepwise deletion experiment, it could be concluded that the first Sp1 site was located between VP-100 and VP-90 and the second Sp1 site was located between VP-90 and VP-80 and that a NRF-1 site spanning between VP-60 and VP-40 could have regulatory effect on VSNL1 promoter activity. To confirm these findings, we introduced point mutations to the sites and tested the mutant constructs in both cell lines (Fig. 3A). Comparable with the 10-bp stepwise deletion results, mutation of two Sp1 sites demonstrated that these sites played a positive regulatory role in VSNL1 transcription activity (Fig. 3, B and C). The putative NRF-1 site in the VSNL1 promoter is an 11-base tandem repeat sequence, GCGCCTGCGCA. When the core CCTGCG was replaced by GCATTC, an 80 and 74% drop in promoter activity was observed as compared with intact VP-60 in NCI-H522 and NCI-H520 cells, respectively. The effect of mutation of the NRF-1-binding site was further verified in a longer VSNL1 promoter setting (VP-174). This mutation caused a 69% decrease of VP-174 activity in NCI-H522 cells and a 44% decrease in NCI-H520 cells. These results suggested that the NRF-1-binding sequence is a very important regulatory element in the VSNL1 promoter.FIGURE 3Deletion and mutation analyses of NRF-1 and Sp1 sites within the VSNL1 gene promoter. A, schematic representation of the VSNL1 promoter and mutant constructs. The filled boxes represent Sp1- and NRF-1-binding sites, respectively. The × symbols on VP-100 and VP-90 indicate where the Sp1 sites were mutated in VP-100 and VP-90. The × symbols on VP-60 and VP-174 indicate where the NRF-1 sites were mutated in VP-60 and VP-174, respectively. B and C, VSNL1 wild type and mutant plasmids were transfected into NCI-H522 and NCI-H520 cells. The luciferase activity was normalized to VP-100 (upper panel) and VP-174 (lower panel). Note that the mutation of NRF-1 site resulted in a pronounced decrease in promoter activity (∼80%) relative to VP-60.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The requirement of NRF-1 for effective VSNL1 promoter activity was verified by co-transfection of the Myc-tagged DN NRF-1 expression vector with VP-174 reporter construct. Overexpression of Myc-DN-NRF-1, containing only the N-terminal DNA-binding and nuclear localization domains, should compete with the binding of endogenous NRF-1 to the NRF-1-binding site on the VSNL1 promoter and therefore interrupt the activating function of endogenous NRF-1 because it does not contain the C-terminal transactivation domain. As shown in Fig. 4(A and B), the VP-174 activity was inhibited upon the expression of Myc-DN-NRF-1, exhibiting ∼64 and 84% inhibition (0.8 μg) in NCI-H522 and NCI-H520 cells, respectively. The expression of Myc-DN-NRF-1 was confirmed by immunoblotting analysis with anti-Myc antibody (Fig. 4C). The second evidence for NRF-1 regulation of the VSNL1 promoter was obtained by NRF-1 siRNA treatment. Transfection of 50 pmol of NRF-1 siRNA resulted in a strong inhibition of NRF-1 protein synthesis as compared with that of scrambled siRNA control (Fig. 5A). When the same amount of NRF-1 siRNA was co-transfected with VP-174, an approximately 90% reduction in promoter activity in both cell lines was observed (Fig. 5B). In addition, the essential requirement of NRF-1 for VILIP-1 transcription was corroborated by the observation that NRF-1 silencing led to a significant down-regulation of VILIP-1 protein (Fig. 5C).FIGURE 5NRF-1 is required for VILIP-1 expression. A, knockdown of NRF-1 expression. NRF-1 siRNA (50 pmol) or the same amount of scrambled control siRNA was transfected, and the cells were lysed in 3 days. NRF-1 was detected with anti-NRF-1 antibody, and equal loading of total protein was demonstrated by Western blot analysis of glyceraldehyde-3-phosphate dehydrogenase. B, VSNL1 promoter activity was suppressed by down-regulation of NRF-1. NRF-1 siRNA (50 pmol) or the same amount of scrambled control siRNA was co-transfected with pGL4.10 VP-174 reporter. Luciferase activity was assayed in 3 days and normalized to scrambled control for both cell lines. C, suppression of VILIP-1 expression by NRF-1 silencing. Either scrambled or NRF-1 siRNA was transfected into NCI-H520 cells, and the expression of NRF-1, VILIP-1, and glyceraldehyde-3-phosphate dehydrogenase was analyzed 3 days post-transfection.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To determine whether the NRF-1 sequence is effectively recognized by cellular NRF-1 protein, we performed EMSA with the nuclear proteins extracted from NCI-H520 cells. Two major bands were observed (Fig. 6A, lane 2) that were abolished by cold (unlabeled) NRF-1 oligonucleotides (lane 3), indicating that these DNA-protein complexes contain proteins able to recognize and bind to the NRF-1 response element. The upper band represented the major NRF-1-containing DNA-protein complex because it was specifically supershifted by using two different NRF-1 antibodies from Drs. Claude Piantadosi and Kimitoshi Kohno, respectively (lanes 4 and 5), whereas the control antibody was unable to supershift it (lane 6). To determine the in vivo occupancy of the VSNL1 promoter by NRF-1, we performed a ChIP assay. As negative controls, a normal rabbit IgG and a nonrelated anti-α1-trypsin antiserum were included for immunoprecipitation using the same batch of cell lysates as used in NRF-1 antibody immunoprecipitation. The VSNL1 promoter was co-immunoprecipitated only with NRF-1 antibody (Fig. 6B), indicating the specific binding of VSNL1 promoter by NRF-1 in vivo. Methylation is one of the major epigenetic mechanisms governing VSNL1 gene expression (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). Methylation of all the CpG islands in VP-1998 with M.SssI treatment nearly abolished VSNL1 promoter activity (decreased to 11%) (11Fu J. Fong K. Bellacosa A. Ross E. Apostolou S. Bassi D.E. Jin F. Zhang J. Cairns P. Ibañez de Caceres I. Braunewell K.H. Klein-Szanto A.J. PLoS ONE. 2008; 3: e1698Crossref PubMed Scopus (26) Google Scholar). Within VP-100, the NRF-1-binding site is the only sequence containing two GCGC sequences. To further study the epigenetic regulation of VSNL1 expressi
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