- Email: [email protected]
NS5ATP9 gene regulated by NF-κB signal pathway
Archives of Biochemistry and Biophysics 479 (2008) 15–19
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
NS5ATP9 gene regulated by NF-jB signal pathway Kang Li a,b, Qingyong Ma a,*, Lei Shi a,b, Chengxue Dang a, Yuan Hong b, Qi Wang b, Yue Li b, Wanhu Fan a,b, Lei Zhang a,b, Jun Cheng b,* a b
The First Hospital of Xi’an Jiaotong University, NO. 277, Yantaxi Road, Xi’an 710061, PR China Infectious Diseases Institute, Beijing Ditan Hospital, 13 Ditan Park, Andingmenwai Dajie, Beijing 100011, PR China
a r t i c l e
i n f o
Article history: Received 23 April 2008 and in revised form 29 July 2008 Available online 14 August 2008 Keywords: NS5ATP9 NF-jB Phage display EMSA DNA-binding protein
a b s t r a c t NS5ATP9 was previously identified as p15PAF [proliferating cell nuclear antigen (PCNA)-associated factor] to bind with PCNA. We earlier identified the promoter region of NS5ATP9 and found NS5ATP9 is a NS5A up-regulation gene. However little is known about how it is regulated. To investigate the gene regulation of NS5ATP9, we screened NS5ATP9 promoter binding proteins using phage display and verified by electrophoretic mobility shift assay (EMSA). We found that the nuclear protein rhNF-jB (p50) could bind to the NS5ATP9 promoter and the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. Our results suggest that NF-jB could participate in the regulation of NS5ATP9 gene expression in carcinogenesis. Ó 2008 Elsevier Inc. All rights reserved.
NS5ATP9 (GenBank Accession No. AF529370) has also been given the names p15PAF, L5, OEACT-1 and KIAA0101. According to the NCBI database, the NS5ATP9 gene is located at 15q22.1 and it consists of 336 bp that encode a 111-residue protein. The gene product of NS5ATP9 was previously found to bind to proliferating cell nuclear antigen (PCNA) in the yeast two-hybrid assay [1]. Recent reports have shown that NS5ATP9 expression is significantly elevated in several types of tumor tissues, and that the gene is involved in the regulation of diverse processes such as DNA repair, apoptosis, cellular signaling pathway and cell growth [1–5]. NS5ATP9 has the oncogenic activity and may be a promising target for development of novel anticancer therapies [2]. One of the most important goals of functional genomics is to compile an interaction map of DNA-binding proteins and their binding sites. The full complement of DNA-binding proteins is responsible for the regulation of gene expression at the transcriptional level [6]. Despite of several studies examining the NS5ATP9 gene, little is known about its binding proteins and how it is regulated. Therefore, it is very important for clarifying NS5ATP9 gene function to investigate for the gene regulation of NS5ATP9. Phage display is a powerful tool for identifying proteins that bind to DNA regulatory elements. Of the screening systems that have been described to date, the most versatile option for building such interaction maps is phage display. Bacteriophages have rela-
tively simple genomes, and they allow the production and screening of large numbers of clones at low-cost. The gel shift assay, or electrophoretic mobility shift assay (EMSA), provides a simple and rapid method for the detection of DNA-binding proteins. This method has been widely used to study sequence-specific DNA-binding proteins such as transcription factors. Fluorescence-based EMSA provides a fast, easy and quantitative method that permits detection of both nucleic acids and proteins in the same gel, thus doubling the amount of information that can be obtained from a single experiment [7]. There is no need to pre-label the DNA or RNA with a radioisotope, biotin, or fluorescent dye before the binding reaction, so there is no possibility that the label will interfere with protein binding [8]. In an earlier study, we showed that NS5ATP9 is up-regulated by hepatitis C virus (HCV) non-structural protein 5A (NS5A) in suppression subtractive hybridization (SSH), and identified the promoter region of the gene [9]. In this study, we screened NS5ATP9 promoter binding proteins using phage display, and we then verified these results by ELISA and EMSA. Our results show that rhNFjB (p50) could bind to the NS5ATP9 promoter and participate in the regulation of NS5ATP9 gene.
Materials and methods Bioinformatic analysis
* Corresponding authors. Fax: +86 029 85323899 (Q. Ma); fax: +86 010 64481639 (J. Cheng). E-mail addresses: [email protected] (Q. Ma), [email protected] (J. Cheng). 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.08.005
In order to analyze the 50 -flanking sequences of the NS5ATP9 gene, we called up the NS5ATP9 genomic sequence from the Ensembl database (http://www.ensembl.org/). We then used the
16
K. Li et al. / Archives of Biochemistry and Biophysics 479 (2008) 15–19
MatInspector software to identify transcription factor binding sites in 2-kbp stretches of the NS5ATP9 promoter sequence (http:// www.genomatix.de/). Amplification of the NS5ATP9 promoter fragments Various sequences: P656 (nucleotides 529 to +127), P288 (nucleotides 161 to +127), P132 (nucleotides 5 to +127) of the 50 -portion upstream of the transcriptional initiation site of NS5ATP9 were amplified by PCR reactions. The PCR primers were as follows: sense primers, P656: 50 -GTA TGT ATA TAC AAT C-30 ; P288: 50 -TGG AGAGTC CTG GTA CC-30 ; P132: 50 -CTC GGG AGA GACCTT GG-30 and antisense primers, 50 -CCT CTC CTC TCT TCT TGT T-30 . All the constructs used in the present study contained an identical 30 -end at base +127, which is contiguous to the NS5ATP9 coding sequence start site, whereas the 50 -end of the constructs varied with the base number indicated in the construct name. Phage display We used P656 (position 529 to +127, relative to the transcription initiation site) to screen for DNA-binding proteins that bind to the NS5ATP9 promoter. The 50 end of the P656 primer was biotinylated to facilitate immobilization of the DNA fragment to multiwell plates. The PCR product was purified using a PCR clean-up kit (Promega, Madison, WI, USA). The T7 Select Human Liver cDNA Library (Novagen) was used for screening. Streptavidin (200 ll, 30 lg/well) was immobilized on the surface of a plastic 24-well NUC plate (Nucleon, Bethesda, USA). The purified NS5ATP9 promoter fragment was diluted in TBS to 1 mg/ml, and 200-ll aliquots were adsorbed to the wells of microtiter plates overnight at 4 °C. The coated wells were blocked using an excess of free biotin overnight at 4 °C to reduce non-specific binding to the streptavidin. Amplified phage library (200 ll) was applied to the ligand-coated plate, and the plate was incubated overnight at 4 °C. The plate was then washed five times with 1 TBST. Phage particles adsorbed to the coated surfaces were eluted by incubation with T7 elution buffer and amplified by infection of E. coli BLT5615 cells. The selection was repeated four times, and the stringency of selection was gradually increased each time. After the final panning cycle, adsorbed phages were cloned by plaque isolation.
DNA fragments (1 lg/well). After washing five times, a blocking step was performed overnight at 4 °C using an excess of free biotin. Phages (5 108 pfu/well) were then added to each well, and the plates were incubated for 2 h at 37 °C. After washing, DNA-phage binding was assayed using a T7 tail fiber monoclonal antibody (1:10,000, Novagen), which was incubated with the plates for one hour at 37 °C. After washing, the plates were incubated for an additional 30 min with an anti-mouse IgG alkaline phosphatase-conjugated antibody (1:20,000, Sigma). The results of the reaction were analyzed at 450 nm using an automated ELISA reader (Bio-Rad, USA). EMSA We used P656, P288 and P132 as the DNA targets in our EMSA assays. PCR was used to generate double stranded (ds) oligonucleotides for the assay. The PCR products were purified using a PCR clean-up kit (Promega). Recombinant human NF-jB (p50) protein (rhNF-jB) and NF-jB consensus oligonucleotide (50 -AGT TGA GGG GAC TTT CCC AGG C-30 ) were purchased from Promega. All of the reactions were loaded onto pre-cast, non-denaturing 4% polyacrylamide gels. The gels containing either DNA, protein, or both DNA and protein were stained using an EMSA kit (Molecular Probes, Eugene, OR, USA). This kit uses two fluorescent dyes for detection, SYBR Green EMSA stain and SYPRO Ruby EMSA stain. The DNA molecular weight marker (100–2000 bp) was obtained from Takara (Dalian, PR China). Results Affinity-based screening of the peptide library using the NS5ATP9 promoter The T7 Select Human Liver cDNA Library was affinity-selected using the NS5ATP9 promoter sequence (P656, Fig. 1). Four cycles of selection/amplification were performed. The enrichment process was monitored by measuring the number of phage recovered. The retained phage were titered after each round of biopanning in order to calculate the rate of recovered phage (retained phage/in-
Determination and analysis of the peptide sequences of the phages that showed binding For PCR analyses, a segment of the phage DNA was amplified by PCR according to the manufacturer’s (Novagen) protocol using the following T7 primers: up, 50 -GGA GCT GTC GTA TTC CAG TC-30 ; down, 50 -AAC CCC TCA AGA CCC GTT TA-30 . The same primers were then used to sequence the DNA products; sequencing reactions were carried out by Invitrogen (Peking). The amino acid sequences of the phage displayed proteins were deduced from the insert sequences. Proteins with homologous sequences were identified by performing a BLAST search in the SwissProt database (http:// www.ncbi.nlm.nih.gov/blast/). The search was conducted using the default parameters, but only Homo sapiens proteins were output in the results. Enzyme-linked immunosorbent assay (ELISA) For phage ELISA, 96-well NUC plates were coated overnight at 4 °C with 100 ll of streptavidin (1 lg/well in PBS buffer). They were then washed five times with 1 PBS containing 0.1% Tween 20, and incubated overnight at 4 °C with 100 ll of biotinylated
Fig. 1. NS5ATP9 promoter sequence fragments. Numbering to the left indicates nucleotide position relative to the major transcription start site, which is designated +1 (Arrowhead). NS5ATP9 coding sequence start site are presented in bold (ATG). Different promoter sequence start sites (529, 161 and 5) are enclosed in boxes. All the constructs contained an identical 30 -end at base +127 (enclosed in box).
K. Li et al. / Archives of Biochemistry and Biophysics 479 (2008) 15–19
17
put phage), which served as the measure of enrichment. Fig. 2 shows that there was a progressive increase in binding efficiency with each cycle of selection/amplification, suggesting that our screening method enriched for specific clones that could bind to the NS5ATP9 promoter. Analysis of peptide sequences
Fig. 2. Rates of phage recovered during four rounds of biopanning. Enrichment of phage during the first two rounds resulted in titer increases of phage recovered in later rounds of biopanning. In rounds three and four, no further increases in recovered phage number were observed, making it unlikely that additional biopanning would lead to further enrichment. Recovered phage rate = retained phage/input phage. Data represent means ± S.D. of three independent experiments.
The amino acid sequences of the phage displayed peptides were deduced from the insert DNA sequences. Several peptide sequences were observed multiple times among the sequenced clones. Pre-screening was carried out by performing colony PCR from a total of 90 random clones from the library, and this showed that the overall cloning efficiency was high. Of these 90 clones, 45 clones (the insert size ranged between 200 and 2000 bp) were randomly sequenced (Fig. 3A). Using the sequences that showed binding in the ELISA (Fig. 3B), we conducted BLAST searches to identify known human homologues. We were able to identify several proteins showing a high degree of homology to the binding sequences; these proteins included both nuclear factors and non-nuclear factors. Since the latter cannot enter the nucleus and regulate gene expression, we focused exclusively on the nuclear factors (Table 1): THOC4, hnRNPR, CLIC1 and P50.
Fig. 3. PCR analysis and ELISA assay. (A) PCR monitoring of insert lengths of the phages selected. DNA markers are shown in the right lane. (B) ELISA reactivity of the phage clones selected by biopanning was performed using a T7 tail fiber monoclonal antibody (1:10000). Individual phage clones were isolated from the titration plate to produce the output phages. A phage clone randomly selected from the initial library was considered a negative control. Data represent means ± S.D. of three independent experiments.
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K. Li et al. / Archives of Biochemistry and Biophysics 479 (2008) 15–19
Table 1 Phage displayed liver proteins that bound to the NS5ATP9 promoter No.
Insert sequence
Homologue of interest
Score
B3
SIQLVTSQIDAQRRPAQSVNRGGMTRNRGAGGF GGGGGTRRGTRGGARGRGRGAGRNSKQQLS AEELDAQLDAYN ARMDTS HDYRGGYEDPYYGYDDGYAVRGRGGGRGGRGAPPPPRGRGAPPPRGRAGYSQRGAPLGPPRGS RGGRGGPAQQQRGRGSRGSRGNRGGNVGGKRKADGYNQPDSKRRQTNNQQNWGSQPIA STCPDDEEIELAYEQVAKALK LTHTIFNPEVFQPQMALPTADGPYLQILEQ
THO complex subunit 4 (Tho4)
82.8
Hetrogeneous nuclear ribonuculeoprotein R (hnRNP R)
115
Chloride intracellular channel protein 1 (CLIC1) Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(P50/P105)
70.2 102
B7 B11 B15
rhNF-jB (p50) binds to the NS5ATP9 promoter To verify P50 binding to the NS5ATP9 promoter, we tested the binding of P50 and the NS5ATP9 promoter fragments (P656, P288 and P132) in EMSA assays. We used PCR to generate ds oligonucleotides encoding the NS5ATP9 promoter target sequence. We titrated these ds NS5ATP9 promoter fragments with increasing
amounts of rhNF-jB (p50), and the titration showed two highmolecular weight bands. The unbound ds fragments migrated in the dye front, whereas the dsDNA-protein complexes were located near the top of the gel. The DNA in the gel was first stained with SYBR Green EMSA DNA stain (Fig. 4A), and then the same gel was stained with SYPRO Ruby EMSA protein stain (Fig. 4B). The pseudocolored images from Fig. 4A, where green represents the
Fig. 4. rhNF-jB (p50) binding to NS5ATP9 promoter. The titration of equal amounts of P656, P288 and P132 with an increasing amount of rhNF-jB (p50) protein was visualized using the AlphaImager. (A) Image of the EMSA gel stained with SYBR Green EMSA DNA stain. P656: Lanes 1 and 7, DNA size markers; Lanes 2–4, 95 ng of P656 with 0, 120 and 240 ng p50; Lane 5, 3.5 pmol NF-jB oligonucleotide and 180 ng P50 as a positive control; Lane 6, free P50 alone as a negative control. P288: Lane 1, DNA size markers; Lane 2, free P50 alone; Lanes 3–5, 44 ng of P288 with 0, 120 and 240 ng p50. P132: Lanes 1–3, 30 ng of P132 with 0, 120 and 240 ng p50; Lane 4 and 5: 44 ng of P288 with 0 and 120 ng p50 as a positive control; Lane 6, DNA size markers. (B) The same gel was stained with SYPRO Ruby EMSA protein stain and destained before taking an image. (C) The images in (A) and (B) were overlaid, and the yellow color represents DNA-protein complexes. Arrows indicate free DNA (For interpretation of color mentioned in this figure, the reader is referred to the web version of this article.).
K. Li et al. / Archives of Biochemistry and Biophysics 479 (2008) 15–19
NS5ATP9 promoter, and Fig. 4B, where red represents rhNF-jB (p50), were overlaid. DNA-protein complexes appear yellow in the overlay (Fig. 4C). The results showed that P50 could bind to P656 and P288, while could not bind to P132, suggesting the binding region contained within 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. Discussion A number of studies have suggested that NS5ATP9 plays a critical role in carcinogenesis, but little is known about how its function is regulated [1,2,5,9]. In this study, the fragment containing the NS5ATP9 promoter region, P656, was used in the phage display approach to identify specific binding proteins. Candidate protein binders were then tested in ELISA. Four different human nuclear proteins were identified as binding to: THOC4, hnRNPR, CLIC1 and rhNF-jB (p50). We then focused on P50 protein and verified the interaction of P50 and the NS5ATP9 promoter by EMSA. And the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. NF-jB also known as Rel/NF-jB is a family of evolutionarily conserved transcription factors involved in responses to environmental changes [10]. Its structure is conserved in species as distinct as sea anemones and humans. The NF-jB family is one of the most-studied eukaryotic transcription factors [11]. There are five members of the mammalian Rel/NF-jB family; p65 (RelA), RelB, c-Rel, p50 (NF-jB1) and p52 (NF-jB2) [12]. p50, a processed product of its p105 precursor [13,14], is the first DNA-binding subunit of the NF-jB transcription factor described [15]. The aminoterminal half of p50 has strong homology to the proto-oncogene, c-rel, its viral counterpart, v-rel and the Drosophila dorsal gene. P50 forms a transcriptionally active heterodimer with NF-jB subunit p65 [13]. Many studies have shown that NF-jB plays an important role in apoptosis, cell-cycle regulation and oncogenesis [16–18]. Enhanced NF-jB activity observed in carcinoma cells is thought to be an important factor in maintaining their survival [19]. This factor has been shown to be elevated in several types of cancer e.g. breast, thyroid, bladder and colon, and several chromosomal alterations involving members of the NF-jB family (cRel, p52 and p65) are located within break point regions in diseases such as non-Hodgkin’s lymphoma and leukemia [12,19]. The previous reports found NS5ATP9 has the oncogenic activity and may be a promising target for development of novel anticancer therapies [2]. The expression of NS5ATP9 is increased in many tumor tissues or cells including esophageal tumor tissue [1], pancreatic cancer cells [2], thyroid carcinoma cells [3] and non-small-cell lung cancer (NSCLC) [4] cells. Exogenous over-expression of NS5ATP9 promoted cancer cell proliferation and transformed NIH3T3 cells in vivo. The silencing of NS5ATP9 significantly suppressed cell growth [2]. In this study, we found that NF-jB (p50), a novel interaction molecular partner of NS5ATP9, could bind to the NS5ATP9 promoter and the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. In fact, besides NF-jB, NS5ATP9 has been shown to interact with a variety of cellular signaling molecules. Our previous study indicated that NS5A can up-regulate the expression of NS5ATP9 gene [9]. The p53–p21 pathway can strictly regulate both the expression and function of NS5ATP9 [2]. Interestingly, NF-jB also associates with NS5A and P53 that interact with NS5ATP9. In chronic hepatitis C, NS5A can modulate the activation of NF-jB [20]. The expression of NS5A in the ER (Endoplasmic reticulum) induces ER stress, ultimately leading to activation of NF-jB [21].
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Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-kB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-kB has been shown to promote resistance to programmed cell death. Crosstalk between the NF-jB and p53 transcription factors can play a pivotal role in determining the cellular response to stress [22,23]. Competition between NF-jB and p53 for CBP (CREB-binding protein) is a crucial determinant of whether a cell proliferates or undergoes apoptosis[24]. Therefore, NS5ATP9 may be regulated by a complex network of interactors, including NF-jB, NS5A and p53 among others. Judging from these results, the function of NS5ATP9 gene may be regulated by NF-jB signal pathway and is critically involved in carcinogenesis. In conclusion, our results show that NF-jB could bind to the NS5ATP9 promoter, and may be associated with functional regulation of the NS5ATP9 gene in carcinogenesis. Acknowledgments We thank our colleagues in the laboratories of the Infectious Diseases Institute of Beijing Ditan Hospital for their advice and help. This work was supported in part by Grants from the National Natural Science Foundation of China (Nos. C39970674 and C03011402). References [1] P. Yu, B. Huang, M. Shen, C. Lau, E. Chan, J. Michel, Y. Xiong, D.G. Payan, Y. Luo, Oncogene 20 (2001) 484–489. [2] M. Hosokawa, A. Takehara, K. Matsuda, H. Eguchi, H. Ohigashi, O. Ishikawa, Y. Shinomura, K. Imai, Y. Nakamura, H. Nakagawa, Cancer Research 67 (2007) 2568–2576. [3] K. Mizutani, M. Onda, S. Asaka, J. Akaishi, S. Miyamoto, A. Yoshida, M. Nagahama, K. Ito, M. Emi, Cancer 103 (2005) 1785–1790. [4] J. Petroziello, A. Yamane, L. Westendorf, M. Thompson, C. McDonagh, C. Cerveny, C.L. Law, A. Wahl, P. Carter, Oncogene 23 (2004) 7734–7745. [5] F. Simpson, K. Lammerts van Bueren, N. Butterfield, J.S. Bennetts, J. Bowles, C. Adolphe, L.A. Simms, J. Young, M.D. Walsh, B. Leggett, L.F. Fowles, C. Wicking, Experimental Cell Research 312 (2006) 73–85. [6] S. Zozulya, M. Lioubin, R.J. Hill, C. Abram, M.L. Gishizky, Nature Biotechnology 17 (1999) 1193–1198. [7] D. Jing, J. Agnew, W.F. Patton, J. Hendrickson, J.M. Beechem, Proteomics 3 (2003) 1172–1180. [8] D. Jing, J.M. Beechem, W.F. Patton, Electrophoresis 25 (2004) 2439–2446. [9] L. Shi, S.L. Zhang, K. Li, Y. Hong, Q. Wang, Y. Li, J. Guo, W.H. Fan, L. Zhang, J. Cheng, Cancer Letters 259 (2008) 192–197. [10] M.S. Hayden, S. Ghosh, Genes & Development 18 (2004) 2195–2224. [11] L.L. Yates, D.C. Gorecki, Acta Biochimica Polonica 53 (2006) 651–662. [12] F. Chen, V. Castranova, X. Shi, The American Journal of Pathology 159 (2001) 387–397. [13] S. Ghosh, A.M. Gifford, L.R. Riviere, P. Tempst, G.P. Nolan, D. Baltimore, Cell 62 (1990) 1019–1029. [14] M. Kieran, V. Blank, F. Logeat, J. Vandekerckhove, F. Lottspeich, O. Le Bail, M.B. Urban, P. Kourilsky, P.A. Baeuerle, A. Israel, Cell 62 (1990) 1007–1018. [15] P.A. Baeuerle, D. Baltimore, Genes & Development 3 (1989) 1689–1698. [16] R. Eldor, A. Yeffet, K. Baum, V. Doviner, D. Amar, Y. Ben-Neriah, G. Christofori, A. Peled, J.C. Carel, C. Boitard, T. Klein, P. Serup, D.L. Eizirik, D. Melloul, Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 5072–5077. [17] S. Shishodia, B.B. Aggarwal, Journal of Biochemistry and Molecular Biology 35 (2002) 28–40. [18] T.D. Gilmore, M. Koedood, K.A. Piffat, D.W. White, Oncogene 13 (1996) 1367– 1378. [19] X. Dolcet, D. Llobet, J. Pallares, X. Matias-Guiu, Virchows Archiv: An International Journal of Pathology 446 (2005) 475–482. [20] I. Mozer-Lisewska, M. Kaczmarek, J. Zeromski, Postepy Biochemii 52 (2006) 56–61. [21] G. Waris, K.D. Tardif, A. Siddiqui, Biochemical Pharmacology 64 (2002) 1425– 1430. [22] S. Rocha, M.D. Garrett, K.J. Campbell, K. Schumm, N.D. Perkins, The EMBO Journal 24 (2005) 1157–1169. [23] G.A. Webster, N.D. Perkins, Molecular and Cellular Biology 19 (1999) 3485– 3495. [24] W.C. Huang, T.K. Ju, M.C. Hung, C.C. Chen, Molecular Cell 26 (2007) 75–87.
Contents lists available at ScienceDirect
Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
NS5ATP9 gene regulated by NF-jB signal pathway Kang Li a,b, Qingyong Ma a,*, Lei Shi a,b, Chengxue Dang a, Yuan Hong b, Qi Wang b, Yue Li b, Wanhu Fan a,b, Lei Zhang a,b, Jun Cheng b,* a b
The First Hospital of Xi’an Jiaotong University, NO. 277, Yantaxi Road, Xi’an 710061, PR China Infectious Diseases Institute, Beijing Ditan Hospital, 13 Ditan Park, Andingmenwai Dajie, Beijing 100011, PR China
a r t i c l e
i n f o
Article history: Received 23 April 2008 and in revised form 29 July 2008 Available online 14 August 2008 Keywords: NS5ATP9 NF-jB Phage display EMSA DNA-binding protein
a b s t r a c t NS5ATP9 was previously identified as p15PAF [proliferating cell nuclear antigen (PCNA)-associated factor] to bind with PCNA. We earlier identified the promoter region of NS5ATP9 and found NS5ATP9 is a NS5A up-regulation gene. However little is known about how it is regulated. To investigate the gene regulation of NS5ATP9, we screened NS5ATP9 promoter binding proteins using phage display and verified by electrophoretic mobility shift assay (EMSA). We found that the nuclear protein rhNF-jB (p50) could bind to the NS5ATP9 promoter and the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. Our results suggest that NF-jB could participate in the regulation of NS5ATP9 gene expression in carcinogenesis. Ó 2008 Elsevier Inc. All rights reserved.
NS5ATP9 (GenBank Accession No. AF529370) has also been given the names p15PAF, L5, OEACT-1 and KIAA0101. According to the NCBI database, the NS5ATP9 gene is located at 15q22.1 and it consists of 336 bp that encode a 111-residue protein. The gene product of NS5ATP9 was previously found to bind to proliferating cell nuclear antigen (PCNA) in the yeast two-hybrid assay [1]. Recent reports have shown that NS5ATP9 expression is significantly elevated in several types of tumor tissues, and that the gene is involved in the regulation of diverse processes such as DNA repair, apoptosis, cellular signaling pathway and cell growth [1–5]. NS5ATP9 has the oncogenic activity and may be a promising target for development of novel anticancer therapies [2]. One of the most important goals of functional genomics is to compile an interaction map of DNA-binding proteins and their binding sites. The full complement of DNA-binding proteins is responsible for the regulation of gene expression at the transcriptional level [6]. Despite of several studies examining the NS5ATP9 gene, little is known about its binding proteins and how it is regulated. Therefore, it is very important for clarifying NS5ATP9 gene function to investigate for the gene regulation of NS5ATP9. Phage display is a powerful tool for identifying proteins that bind to DNA regulatory elements. Of the screening systems that have been described to date, the most versatile option for building such interaction maps is phage display. Bacteriophages have rela-
tively simple genomes, and they allow the production and screening of large numbers of clones at low-cost. The gel shift assay, or electrophoretic mobility shift assay (EMSA), provides a simple and rapid method for the detection of DNA-binding proteins. This method has been widely used to study sequence-specific DNA-binding proteins such as transcription factors. Fluorescence-based EMSA provides a fast, easy and quantitative method that permits detection of both nucleic acids and proteins in the same gel, thus doubling the amount of information that can be obtained from a single experiment [7]. There is no need to pre-label the DNA or RNA with a radioisotope, biotin, or fluorescent dye before the binding reaction, so there is no possibility that the label will interfere with protein binding [8]. In an earlier study, we showed that NS5ATP9 is up-regulated by hepatitis C virus (HCV) non-structural protein 5A (NS5A) in suppression subtractive hybridization (SSH), and identified the promoter region of the gene [9]. In this study, we screened NS5ATP9 promoter binding proteins using phage display, and we then verified these results by ELISA and EMSA. Our results show that rhNFjB (p50) could bind to the NS5ATP9 promoter and participate in the regulation of NS5ATP9 gene.
Materials and methods Bioinformatic analysis
* Corresponding authors. Fax: +86 029 85323899 (Q. Ma); fax: +86 010 64481639 (J. Cheng). E-mail addresses: [email protected] (Q. Ma), [email protected] (J. Cheng). 0003-9861/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2008.08.005
In order to analyze the 50 -flanking sequences of the NS5ATP9 gene, we called up the NS5ATP9 genomic sequence from the Ensembl database (http://www.ensembl.org/). We then used the
16
K. Li et al. / Archives of Biochemistry and Biophysics 479 (2008) 15–19
MatInspector software to identify transcription factor binding sites in 2-kbp stretches of the NS5ATP9 promoter sequence (http:// www.genomatix.de/). Amplification of the NS5ATP9 promoter fragments Various sequences: P656 (nucleotides 529 to +127), P288 (nucleotides 161 to +127), P132 (nucleotides 5 to +127) of the 50 -portion upstream of the transcriptional initiation site of NS5ATP9 were amplified by PCR reactions. The PCR primers were as follows: sense primers, P656: 50 -GTA TGT ATA TAC AAT C-30 ; P288: 50 -TGG AGAGTC CTG GTA CC-30 ; P132: 50 -CTC GGG AGA GACCTT GG-30 and antisense primers, 50 -CCT CTC CTC TCT TCT TGT T-30 . All the constructs used in the present study contained an identical 30 -end at base +127, which is contiguous to the NS5ATP9 coding sequence start site, whereas the 50 -end of the constructs varied with the base number indicated in the construct name. Phage display We used P656 (position 529 to +127, relative to the transcription initiation site) to screen for DNA-binding proteins that bind to the NS5ATP9 promoter. The 50 end of the P656 primer was biotinylated to facilitate immobilization of the DNA fragment to multiwell plates. The PCR product was purified using a PCR clean-up kit (Promega, Madison, WI, USA). The T7 Select Human Liver cDNA Library (Novagen) was used for screening. Streptavidin (200 ll, 30 lg/well) was immobilized on the surface of a plastic 24-well NUC plate (Nucleon, Bethesda, USA). The purified NS5ATP9 promoter fragment was diluted in TBS to 1 mg/ml, and 200-ll aliquots were adsorbed to the wells of microtiter plates overnight at 4 °C. The coated wells were blocked using an excess of free biotin overnight at 4 °C to reduce non-specific binding to the streptavidin. Amplified phage library (200 ll) was applied to the ligand-coated plate, and the plate was incubated overnight at 4 °C. The plate was then washed five times with 1 TBST. Phage particles adsorbed to the coated surfaces were eluted by incubation with T7 elution buffer and amplified by infection of E. coli BLT5615 cells. The selection was repeated four times, and the stringency of selection was gradually increased each time. After the final panning cycle, adsorbed phages were cloned by plaque isolation.
DNA fragments (1 lg/well). After washing five times, a blocking step was performed overnight at 4 °C using an excess of free biotin. Phages (5 108 pfu/well) were then added to each well, and the plates were incubated for 2 h at 37 °C. After washing, DNA-phage binding was assayed using a T7 tail fiber monoclonal antibody (1:10,000, Novagen), which was incubated with the plates for one hour at 37 °C. After washing, the plates were incubated for an additional 30 min with an anti-mouse IgG alkaline phosphatase-conjugated antibody (1:20,000, Sigma). The results of the reaction were analyzed at 450 nm using an automated ELISA reader (Bio-Rad, USA). EMSA We used P656, P288 and P132 as the DNA targets in our EMSA assays. PCR was used to generate double stranded (ds) oligonucleotides for the assay. The PCR products were purified using a PCR clean-up kit (Promega). Recombinant human NF-jB (p50) protein (rhNF-jB) and NF-jB consensus oligonucleotide (50 -AGT TGA GGG GAC TTT CCC AGG C-30 ) were purchased from Promega. All of the reactions were loaded onto pre-cast, non-denaturing 4% polyacrylamide gels. The gels containing either DNA, protein, or both DNA and protein were stained using an EMSA kit (Molecular Probes, Eugene, OR, USA). This kit uses two fluorescent dyes for detection, SYBR Green EMSA stain and SYPRO Ruby EMSA stain. The DNA molecular weight marker (100–2000 bp) was obtained from Takara (Dalian, PR China). Results Affinity-based screening of the peptide library using the NS5ATP9 promoter The T7 Select Human Liver cDNA Library was affinity-selected using the NS5ATP9 promoter sequence (P656, Fig. 1). Four cycles of selection/amplification were performed. The enrichment process was monitored by measuring the number of phage recovered. The retained phage were titered after each round of biopanning in order to calculate the rate of recovered phage (retained phage/in-
Determination and analysis of the peptide sequences of the phages that showed binding For PCR analyses, a segment of the phage DNA was amplified by PCR according to the manufacturer’s (Novagen) protocol using the following T7 primers: up, 50 -GGA GCT GTC GTA TTC CAG TC-30 ; down, 50 -AAC CCC TCA AGA CCC GTT TA-30 . The same primers were then used to sequence the DNA products; sequencing reactions were carried out by Invitrogen (Peking). The amino acid sequences of the phage displayed proteins were deduced from the insert sequences. Proteins with homologous sequences were identified by performing a BLAST search in the SwissProt database (http:// www.ncbi.nlm.nih.gov/blast/). The search was conducted using the default parameters, but only Homo sapiens proteins were output in the results. Enzyme-linked immunosorbent assay (ELISA) For phage ELISA, 96-well NUC plates were coated overnight at 4 °C with 100 ll of streptavidin (1 lg/well in PBS buffer). They were then washed five times with 1 PBS containing 0.1% Tween 20, and incubated overnight at 4 °C with 100 ll of biotinylated
Fig. 1. NS5ATP9 promoter sequence fragments. Numbering to the left indicates nucleotide position relative to the major transcription start site, which is designated +1 (Arrowhead). NS5ATP9 coding sequence start site are presented in bold (ATG). Different promoter sequence start sites (529, 161 and 5) are enclosed in boxes. All the constructs contained an identical 30 -end at base +127 (enclosed in box).
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put phage), which served as the measure of enrichment. Fig. 2 shows that there was a progressive increase in binding efficiency with each cycle of selection/amplification, suggesting that our screening method enriched for specific clones that could bind to the NS5ATP9 promoter. Analysis of peptide sequences
Fig. 2. Rates of phage recovered during four rounds of biopanning. Enrichment of phage during the first two rounds resulted in titer increases of phage recovered in later rounds of biopanning. In rounds three and four, no further increases in recovered phage number were observed, making it unlikely that additional biopanning would lead to further enrichment. Recovered phage rate = retained phage/input phage. Data represent means ± S.D. of three independent experiments.
The amino acid sequences of the phage displayed peptides were deduced from the insert DNA sequences. Several peptide sequences were observed multiple times among the sequenced clones. Pre-screening was carried out by performing colony PCR from a total of 90 random clones from the library, and this showed that the overall cloning efficiency was high. Of these 90 clones, 45 clones (the insert size ranged between 200 and 2000 bp) were randomly sequenced (Fig. 3A). Using the sequences that showed binding in the ELISA (Fig. 3B), we conducted BLAST searches to identify known human homologues. We were able to identify several proteins showing a high degree of homology to the binding sequences; these proteins included both nuclear factors and non-nuclear factors. Since the latter cannot enter the nucleus and regulate gene expression, we focused exclusively on the nuclear factors (Table 1): THOC4, hnRNPR, CLIC1 and P50.
Fig. 3. PCR analysis and ELISA assay. (A) PCR monitoring of insert lengths of the phages selected. DNA markers are shown in the right lane. (B) ELISA reactivity of the phage clones selected by biopanning was performed using a T7 tail fiber monoclonal antibody (1:10000). Individual phage clones were isolated from the titration plate to produce the output phages. A phage clone randomly selected from the initial library was considered a negative control. Data represent means ± S.D. of three independent experiments.
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Table 1 Phage displayed liver proteins that bound to the NS5ATP9 promoter No.
Insert sequence
Homologue of interest
Score
B3
SIQLVTSQIDAQRRPAQSVNRGGMTRNRGAGGF GGGGGTRRGTRGGARGRGRGAGRNSKQQLS AEELDAQLDAYN ARMDTS HDYRGGYEDPYYGYDDGYAVRGRGGGRGGRGAPPPPRGRGAPPPRGRAGYSQRGAPLGPPRGS RGGRGGPAQQQRGRGSRGSRGNRGGNVGGKRKADGYNQPDSKRRQTNNQQNWGSQPIA STCPDDEEIELAYEQVAKALK LTHTIFNPEVFQPQMALPTADGPYLQILEQ
THO complex subunit 4 (Tho4)
82.8
Hetrogeneous nuclear ribonuculeoprotein R (hnRNP R)
115
Chloride intracellular channel protein 1 (CLIC1) Nuclear factor of kappa light polypeptide gene enhancer in B-cells 1(P50/P105)
70.2 102
B7 B11 B15
rhNF-jB (p50) binds to the NS5ATP9 promoter To verify P50 binding to the NS5ATP9 promoter, we tested the binding of P50 and the NS5ATP9 promoter fragments (P656, P288 and P132) in EMSA assays. We used PCR to generate ds oligonucleotides encoding the NS5ATP9 promoter target sequence. We titrated these ds NS5ATP9 promoter fragments with increasing
amounts of rhNF-jB (p50), and the titration showed two highmolecular weight bands. The unbound ds fragments migrated in the dye front, whereas the dsDNA-protein complexes were located near the top of the gel. The DNA in the gel was first stained with SYBR Green EMSA DNA stain (Fig. 4A), and then the same gel was stained with SYPRO Ruby EMSA protein stain (Fig. 4B). The pseudocolored images from Fig. 4A, where green represents the
Fig. 4. rhNF-jB (p50) binding to NS5ATP9 promoter. The titration of equal amounts of P656, P288 and P132 with an increasing amount of rhNF-jB (p50) protein was visualized using the AlphaImager. (A) Image of the EMSA gel stained with SYBR Green EMSA DNA stain. P656: Lanes 1 and 7, DNA size markers; Lanes 2–4, 95 ng of P656 with 0, 120 and 240 ng p50; Lane 5, 3.5 pmol NF-jB oligonucleotide and 180 ng P50 as a positive control; Lane 6, free P50 alone as a negative control. P288: Lane 1, DNA size markers; Lane 2, free P50 alone; Lanes 3–5, 44 ng of P288 with 0, 120 and 240 ng p50. P132: Lanes 1–3, 30 ng of P132 with 0, 120 and 240 ng p50; Lane 4 and 5: 44 ng of P288 with 0 and 120 ng p50 as a positive control; Lane 6, DNA size markers. (B) The same gel was stained with SYPRO Ruby EMSA protein stain and destained before taking an image. (C) The images in (A) and (B) were overlaid, and the yellow color represents DNA-protein complexes. Arrows indicate free DNA (For interpretation of color mentioned in this figure, the reader is referred to the web version of this article.).
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NS5ATP9 promoter, and Fig. 4B, where red represents rhNF-jB (p50), were overlaid. DNA-protein complexes appear yellow in the overlay (Fig. 4C). The results showed that P50 could bind to P656 and P288, while could not bind to P132, suggesting the binding region contained within 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. Discussion A number of studies have suggested that NS5ATP9 plays a critical role in carcinogenesis, but little is known about how its function is regulated [1,2,5,9]. In this study, the fragment containing the NS5ATP9 promoter region, P656, was used in the phage display approach to identify specific binding proteins. Candidate protein binders were then tested in ELISA. Four different human nuclear proteins were identified as binding to: THOC4, hnRNPR, CLIC1 and rhNF-jB (p50). We then focused on P50 protein and verified the interaction of P50 and the NS5ATP9 promoter by EMSA. And the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. NF-jB also known as Rel/NF-jB is a family of evolutionarily conserved transcription factors involved in responses to environmental changes [10]. Its structure is conserved in species as distinct as sea anemones and humans. The NF-jB family is one of the most-studied eukaryotic transcription factors [11]. There are five members of the mammalian Rel/NF-jB family; p65 (RelA), RelB, c-Rel, p50 (NF-jB1) and p52 (NF-jB2) [12]. p50, a processed product of its p105 precursor [13,14], is the first DNA-binding subunit of the NF-jB transcription factor described [15]. The aminoterminal half of p50 has strong homology to the proto-oncogene, c-rel, its viral counterpart, v-rel and the Drosophila dorsal gene. P50 forms a transcriptionally active heterodimer with NF-jB subunit p65 [13]. Many studies have shown that NF-jB plays an important role in apoptosis, cell-cycle regulation and oncogenesis [16–18]. Enhanced NF-jB activity observed in carcinoma cells is thought to be an important factor in maintaining their survival [19]. This factor has been shown to be elevated in several types of cancer e.g. breast, thyroid, bladder and colon, and several chromosomal alterations involving members of the NF-jB family (cRel, p52 and p65) are located within break point regions in diseases such as non-Hodgkin’s lymphoma and leukemia [12,19]. The previous reports found NS5ATP9 has the oncogenic activity and may be a promising target for development of novel anticancer therapies [2]. The expression of NS5ATP9 is increased in many tumor tissues or cells including esophageal tumor tissue [1], pancreatic cancer cells [2], thyroid carcinoma cells [3] and non-small-cell lung cancer (NSCLC) [4] cells. Exogenous over-expression of NS5ATP9 promoted cancer cell proliferation and transformed NIH3T3 cells in vivo. The silencing of NS5ATP9 significantly suppressed cell growth [2]. In this study, we found that NF-jB (p50), a novel interaction molecular partner of NS5ATP9, could bind to the NS5ATP9 promoter and the binding region contained within a 156 bp (nucleotides 5 to 161 bp) immediately upstream of the transcription initiation site. In fact, besides NF-jB, NS5ATP9 has been shown to interact with a variety of cellular signaling molecules. Our previous study indicated that NS5A can up-regulate the expression of NS5ATP9 gene [9]. The p53–p21 pathway can strictly regulate both the expression and function of NS5ATP9 [2]. Interestingly, NF-jB also associates with NS5A and P53 that interact with NS5ATP9. In chronic hepatitis C, NS5A can modulate the activation of NF-jB [20]. The expression of NS5A in the ER (Endoplasmic reticulum) induces ER stress, ultimately leading to activation of NF-jB [21].
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Many cellular stimuli result in the induction of both the tumor suppressor p53 and NF-kB. In contrast to activation of p53, which is associated with the induction of apoptosis, stimulation of NF-kB has been shown to promote resistance to programmed cell death. Crosstalk between the NF-jB and p53 transcription factors can play a pivotal role in determining the cellular response to stress [22,23]. Competition between NF-jB and p53 for CBP (CREB-binding protein) is a crucial determinant of whether a cell proliferates or undergoes apoptosis[24]. Therefore, NS5ATP9 may be regulated by a complex network of interactors, including NF-jB, NS5A and p53 among others. Judging from these results, the function of NS5ATP9 gene may be regulated by NF-jB signal pathway and is critically involved in carcinogenesis. In conclusion, our results show that NF-jB could bind to the NS5ATP9 promoter, and may be associated with functional regulation of the NS5ATP9 gene in carcinogenesis. Acknowledgments We thank our colleagues in the laboratories of the Infectious Diseases Institute of Beijing Ditan Hospital for their advice and help. This work was supported in part by Grants from the National Natural Science Foundation of China (Nos. C39970674 and C03011402). References [1] P. Yu, B. Huang, M. Shen, C. Lau, E. Chan, J. Michel, Y. Xiong, D.G. Payan, Y. Luo, Oncogene 20 (2001) 484–489. [2] M. Hosokawa, A. Takehara, K. Matsuda, H. Eguchi, H. Ohigashi, O. Ishikawa, Y. Shinomura, K. Imai, Y. Nakamura, H. Nakagawa, Cancer Research 67 (2007) 2568–2576. [3] K. Mizutani, M. Onda, S. Asaka, J. Akaishi, S. Miyamoto, A. Yoshida, M. Nagahama, K. Ito, M. Emi, Cancer 103 (2005) 1785–1790. [4] J. Petroziello, A. Yamane, L. Westendorf, M. Thompson, C. McDonagh, C. Cerveny, C.L. Law, A. Wahl, P. Carter, Oncogene 23 (2004) 7734–7745. [5] F. Simpson, K. Lammerts van Bueren, N. Butterfield, J.S. Bennetts, J. Bowles, C. Adolphe, L.A. Simms, J. Young, M.D. Walsh, B. Leggett, L.F. Fowles, C. Wicking, Experimental Cell Research 312 (2006) 73–85. [6] S. Zozulya, M. Lioubin, R.J. Hill, C. Abram, M.L. Gishizky, Nature Biotechnology 17 (1999) 1193–1198. [7] D. Jing, J. Agnew, W.F. Patton, J. Hendrickson, J.M. Beechem, Proteomics 3 (2003) 1172–1180. [8] D. Jing, J.M. Beechem, W.F. Patton, Electrophoresis 25 (2004) 2439–2446. [9] L. Shi, S.L. Zhang, K. Li, Y. Hong, Q. Wang, Y. Li, J. Guo, W.H. Fan, L. Zhang, J. Cheng, Cancer Letters 259 (2008) 192–197. [10] M.S. Hayden, S. Ghosh, Genes & Development 18 (2004) 2195–2224. [11] L.L. Yates, D.C. Gorecki, Acta Biochimica Polonica 53 (2006) 651–662. [12] F. Chen, V. Castranova, X. Shi, The American Journal of Pathology 159 (2001) 387–397. [13] S. Ghosh, A.M. Gifford, L.R. Riviere, P. Tempst, G.P. Nolan, D. Baltimore, Cell 62 (1990) 1019–1029. [14] M. Kieran, V. Blank, F. Logeat, J. Vandekerckhove, F. Lottspeich, O. Le Bail, M.B. Urban, P. Kourilsky, P.A. Baeuerle, A. Israel, Cell 62 (1990) 1007–1018. [15] P.A. Baeuerle, D. Baltimore, Genes & Development 3 (1989) 1689–1698. [16] R. Eldor, A. Yeffet, K. Baum, V. Doviner, D. Amar, Y. Ben-Neriah, G. Christofori, A. Peled, J.C. Carel, C. Boitard, T. Klein, P. Serup, D.L. Eizirik, D. Melloul, Proceedings of the National Academy of Sciences of the United States of America 103 (2006) 5072–5077. [17] S. Shishodia, B.B. Aggarwal, Journal of Biochemistry and Molecular Biology 35 (2002) 28–40. [18] T.D. Gilmore, M. Koedood, K.A. Piffat, D.W. White, Oncogene 13 (1996) 1367– 1378. [19] X. Dolcet, D. Llobet, J. Pallares, X. Matias-Guiu, Virchows Archiv: An International Journal of Pathology 446 (2005) 475–482. [20] I. Mozer-Lisewska, M. Kaczmarek, J. Zeromski, Postepy Biochemii 52 (2006) 56–61. [21] G. Waris, K.D. Tardif, A. Siddiqui, Biochemical Pharmacology 64 (2002) 1425– 1430. [22] S. Rocha, M.D. Garrett, K.J. Campbell, K. Schumm, N.D. Perkins, The EMBO Journal 24 (2005) 1157–1169. [23] G.A. Webster, N.D. Perkins, Molecular and Cellular Biology 19 (1999) 3485– 3495. [24] W.C. Huang, T.K. Ju, M.C. Hung, C.C. Chen, Molecular Cell 26 (2007) 75–87.