Báo cáo khoa học: Cloning and functional analysis of 5¢-upstream region of the Pokemon gene pptx

Cloning and functional analysis of 5¢-upstream region of the Pokemon gene Yutao Yang, Xiaowei Zhou, Xudong Zhu, Chuanfu Zhang, Zhixin Yang, Long Xu and Peitang Huang Laboratory of Protein Engineering, Beijing Institute of Biotechnology, China The poxvirus and zinc finger (POZ) domain, formerly termed the broad complex, tramtrack and bric-a-brac (BTB) domain, was initially characterized in the Drosophila proteins broad complex, tramtrack and bric-a-brac [1]. It is  120 amino acids long and usu- ally exists in a few transcriptional repression complexes [2]. The POZ domain is highly conserved from yeast to humans, and is involved in many critical cellular pro- cesses such as development [3,4], oncogenesis [5,6], apoptosis [7] and ion channel activity [8]. More than 200 proteins have been found in associa- tion with the POZ domain [9], and they are usually grouped according to their distinct C-terminal struc- tures, such as the zinc finger motif, basic zipper motif, actin-binding repeats, kech domains and ion channel motifs [10]. Proteins containing the POZ domain and zinc finger motif are termed POZ-ZF or POK proteins. Via the POZ domain, many POK proteins can recruit transcriptional co-repressors such as nuclear co-repres- sor (N-CoR), silencing mediator of retinoic acid, thyroid hormone receptor (also known as N-CoR2), mSin3A and histone deacetylases to the target gene promoter regions, thereby decreasing these gene tran- scriptional activities [11–14]. Currently,  60 POK genes have been identified in the human genome [15]. Many of them, such as PLZF, BCL-6, Zbtb7 and HIC1, are involved in development, differentiation and oncogenesis [2]. Pokemon, the POK erythroid myeloid ontogenic factor, was previously known by several names (LRF, OCZF and FBI-1) and was originally identified as a protein that binds specifi- cally to the inducer of short transcripts (IST) element Keywords DNA decoy; element; mutation; Pokemon; promoter Correspondence P. Huang, Laboratory of Protein Engineering, Beijing Institute of Biotechnology, Beijing 100071, China Fax ⁄ Tel: +86 10 6381 0272 E-mail: amms832@126.com (Received 15 October 2007, revised 12 February 2008, accepted 18 February 2008) doi:10.1111/j.1742-4658.2008.06344.x Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expres- sion of the Pokemon gene in humans, its 5¢-upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG-U and NEG-D elements were involved in negative regulation of the Pokemon promoter, whereas the POS-D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG-U, NEG-D and POS-D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG-U and NEG-D elements led to negative regu- lation of the Pokemon promoter. Moreover, the NEG-U and NEG-D ele- ments needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy. Abbreviations BTB, broad complex, tramtrack and bric-a-brac domain; EMSA, electrophoretic mobility shift assay; Pokemon, POK erythroid myeloid ontogenic factor; POZ, poxvirus and zinc finger domain; SRE, sterol regulatory element; SREBP, SRE-binding protein. 1860 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS on the HIV-1 genome [16]. It was first termed the fac- tor binding to IST-1 (FBI-1) and is encoded by the Zbtb7 gene. Pokemon not only regulates the HIV-1 Tat transactivation process [17,18], but is also involved in human and murine adipogenesis [19]. It acts as a transcription factor and regulates the expression of many gene-encoding proteins such as extracellular matrix collagen types I, II, IX, X and XI, fibronectin, elastin, human cartilage oligomeric matrix protein [20,21], ARF tumor suppressor [22], and the c-fos and c-myc oncoproteins [23]. This activity is due to its capacity to bind to the consensus sequence within the promoters of these target genes. Furthermore, Pokemon can regulate the expression of other genes via an inter- action between its POZ domain and other important transcription factors such as Sp-1 and the p65 subunit of NF-jBorIjB [24,25]. Pokemon is also a repressor of the ARF tumor sup- pressor gene and is a central regulator in oncogenesis. Overexpression of the Pokemon gene can decrease expression of the ARF gene, which in turn results in p53 degradation and oncogenic transformation. Con- versely, depletion of the Pokemon gene both inhibited oncogene-mediated cellular transformation and induced cell senescence and apoptosis [22,26]. There- fore, Pokemon plays a crucial role in cell tumorigenesis and may be a potential therapeutic target for human cancer therapy. Although considerable work has been done to eluci- date the biological functions of the Pokemon, its regu- lation mechanism has not been reported. In order to identify the elements that regulate Pokemon gene expression, we cloned and characterized the Pokemon promoter. Our results suggest a role for two strongly negative elements and one positive element in regula- tion of the Pokemon gene. However, the two negative elements could not individually exhibit the negative regulatory activity; they required mutual cooperation with each other in order to negatively regulate the Pokemon promoter. In conclusion, our studies are the first to elucidate transcriptional regulation mechanism of the Pokemon gene, and this will be beneficial for gene therapy in cancer. Results Cloning of the 5¢-upstream region of the Pokemon gene To identify the regulatory sequences that control expression of the Pokemon gene, a 2204-bp section of the 5¢-upstream region of the Pokemon gene was cloned by PCR using human genomic DNA as the template. Figure 1 shows the nucleotide sequence of the 2204-bp promoter region and a short stretch of the transcription region. The translation start site was des- ignated as +1, and the transcribed region was shaded. Some reports showed that the Pokemon gene can be expressed in different cell lines and different human tis- sues [25,27]; later reports also confirmed these results [22,26]. To examine whether the Pokemon promoter can drive reporter gene expression in a similar manner, the 2204-bp promoter linked to the luciferase reporter gene was used in transient transfection studies with dif- ferent cell lines. Luciferase assays showed that the Pokemon promoter could direct luciferase expression in HeLa, A549, DU145, Jurkat and HepG2 cells, whereas the pGL3-basic construct could not (Fig. 2); this suggests that the Pokemon promoter can drive reporter gene expression in different cell lines, which was in agreement with the expression patterns of the Pokemon gene [22,25–27]. Because Pokemon is also highly expressed in lung and prostate carcinomas, A549 and DU145 cells were used to study the regula- tion mechanisms of the Pokemon gene. Computer analysis of putative transcription factor-binding sites For a rough understanding of the regulation of the Pokemon gene, the 2204-bp section of its 5¢-upstream region was analyzed for putative cis-elements in the TRANSFAC 7.0 database (http://www.generegulation. com/pub/databases.html) [28]. After scanning the TRANSFAC 7.0 database, we found that the putative TATA and CCAAT sequences are absent in the upstream region of the Pokemon gene; however, some transcription factor-binding sequences, including Sp1, AP-1, AP-2, PU.1, Hb, CBF-1, GATA-1 elements and p53-binding sites are present in the promoter (Fig. 1), implying their potential roles in the regulation of the Pokemon gene. Deletion analysis of the Pokemon promoter To broadly determine the main regulatory regions in the Pokemon promoter, we created five 5¢-deletion con- structs; the activities of these deletion constructs were measured in A549 and DU145 cells. As shown in Fig. 3A, luciferase activity was markedly reduced when the region from )837 to )560 was deleted, but was dramatically increased when the region from )560 to )233 was deleted. These results demonstrated the pos- sible presence of some potential positive elements in the region from )837 to )560 and negative elements in the region from )560 to )233. Y. Yang et al. Analysis of upstream region of the Pokemon gene FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS 1861 To determine the regulatory region in the Pokemon promoter more accurately, we performed further 5¢-deletion analysis with the regions from )837 to )560 and )560 to )233. Five 5¢-deletion constructs were constructed for the region from )837 to )560 and used in transient transfection studies. As shown in Fig. 3B, only the deletion from )580 to )560 resulted in a moderate reduction in luciferase activity; it reduced the luciferase activity of A549 cells by 4.6-fold and that of DU145 cells by 4.2-fold compared with Fig. 1. Nucleotide sequence of the 5¢-upstream region of the Pokemon gene. The upstream region of the Pokemon gene containing the pro- moter and a short stretch of the transcribed region is shown. The nucleotides are numbered on the left, with the translation start site desig- nated as +1. The translation start site is indicated by an arrowhead. The transcribed region is shaded. The POS-D, NEG-U and NEG-D elements are boxed. The putative cis-elements are underlined. The TRANSFAC database was used to identify putative cis-elements in the 5¢-upstream region of the Pokemon gene. Analysis of upstream region of the Pokemon gene Y. Yang et al. 1862 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS F-580, suggesting the presence of potential positive element(s) in this region. Six 5¢-deletion constructs were constructed for the region from )560 to )233, and their activities were measured in A549 and DU145 cells. Figure 3C shows that deletion of the region from )560 to )542 resulted in a remarkable increase in luciferase activity, by 25-fold in A549 cells and 24.6-fold in DU145 cells compared with F-560, sug- gesting the presence of a strong negative element in this region. This negative element was termed NEG-U. The F-233 construct still directed reporter gene expression to a great degree, and some essential ele- ments might be responsible for this property. Further deletion analysis showed that the region from )83 to )71 was responsible for the strong activity of the 0 1 2 3 4 5 6 7 8 9 HeLa A549 DU145 Jurkat HepG2 LUC activity Fig. 2. The 2204-bp section of the Pokemon promoter can drive luciferase gene expression in different cell lines. Different cell lines were transfected with the 2204-bp section of the Pokemon promoter construct or the pGL3-basic construct. Solid bars represent the 2204-bp stretch showing Pokemon promoter construct activity, and open bars represent pGL3-basic construct activity. The values are the mean ± SE for three independent experiments performed in triplicate and are normalized to Renilla luciferase activity. A B C D LUC activity LUC activity LUC activity LUC activit y Fig. 3. 5¢-Deletion analysis of the Pokemon promoter. Progres- sively truncated fragments of the upstream region of the Pokemon gene were inserted into the pGL3-basic vector and their ability to activate transcription of the luciferase gene was assessed in A549 and DU145 cells. The values are the mean ± SE for three indepen- dent experiments performed in triplicate and are normalized to Renilla luciferase activity. (A) Rough characterization of the Poke- mon promoter using the larger, gradually truncated fragment from )2220 to )233. (B) Refined analysis of the Pokemon promoter using the smaller, progressively truncated fragment from )837 to )560. (C) Refined analysis of the Pokemon promoter using the smaller, progressively truncated fragment from )560 to )233. (D) Refined analysis of the Pokemon promoter using the smaller, progressively truncated fragment from )233 to )71. Y. Yang et al. Analysis of upstream region of the Pokemon gene FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS 1863 Pokemon promoter. When this region was removed, luciferase activity was decreased by 25.6-fold in the A549 cells and 23.4-fold in the DU145 cells compared with F-83, indicating that the region from )83 to )71 is necessary for strong expression of the Pokemon pro- moter in both A549 and DU145 cells (Fig. 3D); we termed this positive element as POS-D. Importance of the POS-D element for the strong activity of the Pokemon promoter Because the POS-D element plays an important role in the strong activity of the Pokemon promoter, it may be the target of some transcriptional factors. To deter- mine the presence of binding sites for transcriptional factors in this element, we performed electrophoretic mobility shift assays (EMSAs) with A549 cell nuclear extract. Figure 4A shows the formation of complexes when wild-type double POS-D was used as a probe and incubated with the nuclear extracts. The specificity of the complexes was confirmed by incubation with a 50-fold excess of unlabeled wild-type double POS-D. However, mutant POS-D did not compete with the labeled wild-type probe, suggesting that the POS-D element is specifically recognized by nuclear proteins from A549 cells. Because the POS-D element is responsible for the strong activity of the Pokemon promoter, we specu- lated whether mutation of the POS-D element would result in a decrease in the activity of the promoter. We mutated a 9-bp section of the POS-D element in the F-233 construct (MF-233) and transfected MF-233 and F-233 into A549 and DU145 cells, respectively. Luciferase assays showed that MF-233 displayed lower luciferase activity than F-233 (Fig. 4B). In addition, we also examined the function of the POS-D element by using the DNA decoy technique. Our results showed that introduction of the POS-D decoy could efficiently suppress the F-233 activity, whereas the mutant POS-D decoy could not (Fig. 4C). All these results suggest that POS-D is an essential regulatory element that is responsible for the strong activity of the Pokemon promoter. Role of the NEG-U element in the negative regulation of the Pokemon promoter 5¢-Deletion analysis showed that the NEG-U element was involved in the negative regulation of the Pokemon A B C Fig. 4. The POS-D element is necessary for strong activity of the Pokemon promoter. (A) EMSA was performed with 32 P-labeled POS-D element in the absence or presence of the wild-type POS-D element or mutant POS-D element at the molar excess indicated above each lane. (B) Activities of F-233 and MF-233 in A549 and DU145 cells. (C) Activities of F-233 and varying amounts of the decoy oligonucleotides in A549 and DU145 cells. WP-decoy indi- cates the wild-type POS-D oligonucleotide, while MP-decoy indi- cates the mutant POS-D oligonucleotide. The values are the mean ± SE for three independent experiments performed in tripli- cate and are normalized to Renilla luciferase activity. Analysis of upstream region of the Pokemon gene Y. Yang et al. 1864 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS promoter. To examine whether the NEG-U element shows nuclear protein-binding activity, we synthesized double NEG-U and mutant double NEG-U elements, and then performed EMSAs with A549 cell nuclear extracts. As shown in Fig. 5A, specific complexes were observed with the labeled wild-type probe; moreover, 250-fold excess of the unlabeled wild-type probe almost entirely eliminated complex formation, whereas 250-fold excess of the unlabeled mutant probe did not. Interestingly, we found that the mutated element can compete with the wild-type NEG-U element to some extent; this suggests that the corresponding nuclear factor may bind to the region between the mutation site and the marginal sequence in the mutant probe. However, our decoy analysis showed that the MNEG-U decoy had almost no effect on the activity of F-560, indicating that the mutated competitor had only weak nonspecific binding capacity for proteins in the A549 nuclear extract (Fig. 5B). Because the NEG-U element lent a strong negative character to the Pokemon promoter, we speculated whether it could also decrease the activity of the SV40 promoter. NEG-U and mutant NEG-U elements were cloned into the KpnI ⁄ XhoI sites of the pGL3-control plasmid in both the normal and reverse orientations, and the resultant constructs were used in transient transfection studies. Interestingly, as shown in Fig. 5C, both normally and reversely oriented NEG-U elements increased the activity of the SV40 promoter, whereas the mutant element did not. Therefore, the NEG-U element could exhibit the negative regulatory function only in a special DNA context. Role of the NEG-D element in the negative regulation of the Pokemon promoter The NEG-U element alone cannot negatively regulate the function of the Pokemon promoter, therefore, we proposed that it might interact with other downstream regulatory elements to exhibit negative activity. To accurately locate the region that can cooperate with the NEG-U element, we performed 3¢-deletion analysis in the region from )560 to )88. All the deletion con- structs of this region contained the region between )88 and )17 but different internal deletion fragments. As B A WM Competitor Nuclear protein Free probe Complex 0 0 50× 250× 50× 250× -+++ ++ C SV40-Promoter LUC SV40-Promoter LUC LUC LUC LUC NEG-U SV40-Promoter MNEG-U SV40-Promoter MNEG-U SV40-PromoterNEG-U 0 50 100 150 MU- I MU- F WU-I WU-F W LUC activit y A549 DU145 F-560 (ng) 300 300 300 300 300 WU-decoy (µg) 0 1 2 0 0 MU-decoy (µg) 0 0 0 1 2 0 1 2 3 4 5 6 LUC acitivity A549 DU145 Fig. 5. The NEG-U element is involved in the negative regulation of the Pokemon promoter. (A) EMSA was performed with 32 P-labeled NEG-U element in the absence or presence of the wild-type NEG-U element or mutant NEG-U element at the molar excess indicated above each lane. (B) Activities of F-560 and varying amounts of the decoy oligonucleotide in A549 and DU145 cells. WU-decoy indi- cates the wild-type NEG-U oligonucleotide, wherreas MU-decoy indicates the mutant NEG-U oligonucleotide. (C) The left-hand panel shows the different chimeric constructs used to test the effect of the NEG-U element on the SV40 promoter. The right-hand panel shows the results of luciferase activity assays for different chimeric constructs in A549 cells and DU145 cells. The values are the mean ± SE for three independent experiments performed in tripli- cate and are normalized to Renilla luciferase activity. Y. Yang et al. Analysis of upstream region of the Pokemon gene FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS 1865 shown in Fig. 6A, deleting the region from )127 to )88 resulted in a significant increase in luciferase activity compared with F-560, whereas deleting the region from )107 to )88 resulted in a slight increase in luciferase activity, indicating that the region from )127 to )107 is also involved in the negative regula- tion of the Pokemon promoter; we termed this region NEG-D. In addition, progressive deletion of the region from )156 to )473 resulted in only slight changes in luciferase activity compared with T-127, further con- firming the importance of the NEG-D element. In order to fully examine the function of the NEG-D ele- ment, we performed EMSA and NEG-D decoy analy- sis. EMSA showed that the NEG-D element could be bound specifically by the nuclear extract from A549 cells (Fig. 6B). Decoy analysis demonstrated that the NEG-D decoy could increase the activity of F-560; a similar observation was made with regard to the NEG-U decoy-treated cells (Fig. 6C). These results indicate that the NEG-D element is also necessary for negative regulation of the Pokemon promoter. Because the NEG-D element also lent a strong nega- tive character to the Pokemon promoter, we speculated whether it might decrease the activity of the SV40 Fig. 6. The NEG-D element is involved in the negative regulation of the Pokemon promoter. (A) The left-hand panel shows 3¢-deletion con- structs in the region between )560 and )88 of the Pokemon promoter. All these constructs contained the region between )88 and )17 of the Pokemon promoter but had different internal deletion fragments. The right-hand panel shows the results of the luciferase activity assays of the 3¢-deletion constructs in A549 and DU145 cells. (B) EMSA was performed with 32 P-labeled NEG-D element in the absence or pres- ence of the wild-type NEG-D element or mutant NEG-D element at the molar excess indicated above each lane. (C) Activities of F-560 and varying amounts of the decoy oligonucleotide in A549 and DU145 cells. WD-decoy indicates the wild-type NEG-D oligonucleotide, whereas MD-decoy indicates the mutant NEG-D oligonucleotide. (D) The left-hand panel shows the different chimeric constructs used to test the effect of the NEG-D element on the SV40 promoter. The right-hand panel shows the results of the luciferase activity assays for different chi- meric constructs in A549 and DU145 cells. The values are the mean ± SE for three independent experiments performed in triplicate and are normalized to Renilla luciferase activity. Analysis of upstream region of the Pokemon gene Y. Yang et al. 1866 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS promoter. NEG-D and mutant NEG-D elements were also cloned into the Kpn I ⁄ XhoI sites of the pGL3-con- trol plasmid in both the normal and reverse orienta- tions, and the activities of the resultant construct were assayed. As shown in Fig. 6D, both normal- and reverse-oriented NEG-D elements increased the activ- ity of the SV40 promoter, whereas the mutant element did not; this is similar to the function of the NEG-U element, suggesting that a single NEG-D element alone cannot exhibit negative activity. The NEG-U element cooperates with the NEG-D element to promote negative regulation of the Pokemon promoter To further characterize the effects of the NEG-U and NEG-D elements on the negative regulation of the Pokemon promoter, mutations of the NEG-U and NEG-D elements, alone or in combination, were cre- ated in F-560 and transiently transfected into A549 and DU145 cells. As shown in Fig. 7A, mutation of the NEG-U element alone resulted in a significant increase in luciferase activity; a similar result was also observed in the construct that only harbored the mutated NEG-D element. Interestingly, mutations in both sites also led to a remarkable increase in lucifer- ase activity. These results indicate that both the NEG-U and NEG-D elements are essential for negative regulation of the Pokemon promoter. To examine the impact of the length between the two elements on inter-region synergism, the effect of deletions in the intervening sequence was evaluated. Our results showed that the inhibition of the synergis- tic activity of the NEG-U and NEG-D elements was almost abolished in D-254 and D-106 (Fig. 7B), thus suggesting that the inhibition of synergism may require the NEG-U and NEG-D elements to be located at a certain appropriate distance from each other. To further determine the cooperation between the NEG-U and NEG-D elements, we performed DNA A B C Fig. 7. The NEG-U and NEG-D elements are necessary for the neg- ative regulation of the Pokemon promoter. (A) The left-hand panel shows the F-560 and mutant constructs. The right-hand panel shows the results of the luciferase activity assays for all the con- structs in A549 and DU145 cells. M-U indicates that the NEG-U ele- ment was mutated in F-560, M-D indicates that the NEG-D element was mutated in F-560 and M-B indicates that both the NEG-U and NEG-D elements were mutated in F-560. (B) The left- hand panel shows F-560 and different mutant constructs harboring shorter intervening sequences (254 and 106 bp) between the NEG- U and NEG-D elements. The distance between the NEG-U and NEG-D elements was 253 and 106 bp in D-254 and D-106, respec- tively. The right-hand panel shows the results of the luciferase activity assays for different constructs in A549 and DU145 cells. (C) The effects of 2 lg of the NEG-U decoy, 2 lg of the NEG-D decoy and a combination of 1 lg of each decoy on the activity of F-560. The open ellipse indicates the wild-type NEG-U element, whereas the solid ellipse indicates the mutant NEG-U element; the open triangle indicates the wild-type NEG-D element, whereas the solid triangle indicates the mutant NEG-D element. ‘U’ indicates the NEG-U decoy, ‘D’ indicates the NEG-D decoy and ‘U+D’ indi- cates the combination of the decoys. The values are the mean ± SE for three independent experiments performed in tripli- cate and are normalized to Renilla luciferase activity. Y. Yang et al. Analysis of upstream region of the Pokemon gene FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS 1867 decoy experiments using 2 lg of the NEG-U decoy, 2 lg of the NEG-D decoy, and a combination of 1 lg each of the NEG-U and NEG-D decoys. Our results demonstrated that the F-560 activity is increased more by the combination of the NEG-U and NEG-D decoys than by the individual NEG-U and NEG-D decoys (Fig. 7C). These data demonstrate that the Pokemon promoter can only be negatively regulated when the NEG-U element cooperates with the NEG-D element. Discussion Pokemon, a member of the POK protein family, plays an important role in cell development, differentiation and oncogenesis. Abrogation of Pokemon often leads to cell-cycle arrest and cellular senescence and apopto- sis. However, overexpression of Pokemon will lead to reduced levels of the tumor suppressor gene ARF, resulting in degradation of the wild-type nuclear p53 and oncogenic transformation [22,26]. Although a con- siderable amount of work has been done characterizing the function of Pokemon, very little is known about the mechanism that governs its expression. In this study, we performed deletion analysis, mutation analy- sis, as well as decoy assays, and found that the NEG-U, NEG-D and POS-D elements play important roles in regulation of the Pokemon promoter; this helps us understand the transcriptional mechanism of the Pokemon gene. In humans, the Pokemon gene localizes in syntenic chromosomal regions (19p13.3), and is widely expressed in adult tissues and cell lines [27]. Reports have shown that alternative splicing and alternative promoters play important roles in the regulation of some genes [29–31]. Interestingly, our previous studies also showed that the Pokemon transcripts could be alternatively spliced, resulting in the formation of mRNAs with four different 5¢-untranslated regions. Matching the nucleotide sequences of four first exons to human genomic DNA showed that four alternative first exons were located at )11 596, )10 224, )9109 and )17 bp upstream of the translation start site of the Pokemon gene, suggesting that the Pokemon gene could be regulated by four alternative promoters. We are currently performing deletion analysis and a DNA decoy assay to study three other alternative promoters, which will further provide better understanding of the Pokemon gene transcriptional mechanisms. From the TRANSFAC 7.0 database, we found some putative regulatory elements in the Pokemon promoter, including binding sites for Sp1, AP-1, AP-2 and GATA-1 elements (Fig. 1); however, deletion analysis showed that the above-mentioned regulatory elements cannot play decisive roles in the regulation of the Pokemon gene, suggesting the complexity of gene regu- lation. Fortunately, we found that three regulatory ele- ments, namely, POS-D, NEG-U and NEG-D, play important roles in the regulation of the Pokemon gene. To determine whether these three elements are homo- logous with the regulatory sequences deposited in the database, we performed a BLAST search by using their sequences as queries in the TRANSFAC 7.0 database. The results showed that none of them shared a higher degree of homology with the reported regula- tory elements, thus indicating their novel roles. Cur- rently, we are conducting yeast one-hybridization in order to isolate transcription factors that can interact with these novel regulatory elements; this will help further understand the regulatory mechanism of the Pokemon promoter. The DNA decoy technique, also referred to as the transcription factor decoy technique, involves the transfection of double-stranded oligodeoxynucleotides corresponding to the regulatory sequence into target cells; this results in the attenuation of authentic cis– trans interactions, leading to the removal of transcrip- tion factors from the endogenous regulatory element and suppression of the expression of the regulated genes. Recently, some reports showed that the DNA decoy technique is a powerful tool for therapy related to various diseases [32–34]. In this experiment, we used the DNA decoy technique to successfully confirm the function of the POS-D, NEG-U and NEG-D elements, proving that the transcription factor decoy technique can be a powerful tool for the study of transcriptional regulation mechanisms. However, we also found that the wild-type POS-D decoy cannot completely abolish reporter gene expression; this may occur for two rea- sons. First, some DNA decoys may be degraded by endogenous nuclease. Second, in the amounts used, the POS-D decoy cannot completely abolish the inter- action between the wild-type POS-D element and its corresponding transcription factor. Although POS-D decoys cannot completely abolish the activity of the Pokemon promoter, they are still potential oligodeoxy- nucleotides that can be used to decrease the Pokemon gene expression in cancer gene therapy. The SV40 early promoter contains a TATA box, three copies of a 21-bp GC-rich repeat, and two copies of a 72-bp repeat. The 72-bp repeat acts as an enhan- cer to increase the activity of the SV40 promoter, whereas the 21-bp GC-rich repeat is the main recogni- tion signal for eukaryotic RNA polymerase II and is necessary for promoter activity [35]. Deletion analysis and the decoy assay showed that the NEG-U and NEG-D elements were involved in the negative Analysis of upstream region of the Pokemon gene Y. Yang et al. 1868 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS regulation of the Pokemon gene. However, our gain- of-function experiment interestingly revealed that both the NEG-U and NEG-D elements could increase the activity of the SV40 promoter. The negative function of the NEG-U element is strictly dependent on the NEG-D element. When incorporated upstream of the SV40 promoter, the NEG-U element may interact with the 72-bp repeat enhancer to increase promoter activ- ity. In addition, there may be a similar reason why NEG-D element could increase the activity of the SV40 promoter. Therefore, a DNA context in which different regulatory elements exist also plays an impor- tant role in gene regulation, and incorporating these elements into new promoters may alter their original functions. Eukaryotic gene expression is often controlled by multiprotein transcriptional complexes that bind differ- ent elements in the 5¢-upstream regions of target genes [36]. Gallagher et al. showed that the GATA-1 and Oct-1 elements were required for the expression of the gene encoding human a-hemoglobin-stabilizing protein [37]. Recently, Griffin et al. showed that E-box and sterol regulatory element (SRE) could mediate syner- gistic activation of the fatty acid synthase promoter [38]. NEG-U and NEG-D elements were necessary and sufficient for the negative regulation of the Pokemon gene, but the NEG-U or NEG-D element alone could not negatively affect gene expression, further confirm- ing the importance of combinatorial control. In this study, we also found that mutations in both NEG-U and NEG-D elements had the same effect as each sin- gle mutation. This is because negative regulation of the Pokemon gene is strictly dependent on an interaction between NEG-U and NEG-D elements. Mutations in both sites or mutation in a single site could abolish an interaction between them. Therefore, all mutated con- structs displayed high luciferase activities. In many eukaryotic genes, transcription factors bind to promoters located at sites distant from one another, yet they act synergistically via DNA looping to acti- vate transcription [39,40]. The insulin gene promoter contains three SREs and two E-boxes; two of the SREs overlap with the E-boxes that can be bound by the BETA2 ⁄ E47 protein. Activation of the insulin pro- moter by SRE-binding protein (SREBP-1c) was mark- edly enhanced by the co-expression of BETA2 ⁄ E47. Synergistic activation by SREBP-1c and BETA2⁄ E47 was not mediated via SREs but via the E-boxes. Reducing the distance between the two E-boxes abol- ished synergistic activation. Therefore, the synergistic action required the presence of two E-boxes separated by an appropriate distance in a looped form, presum- ably to form a DNA and SREBP-1c ⁄ BETA2 ⁄ E47 complex [41]. To determine whether the length between the NEG-U and NEG-D elements also plays an important role in the regulation of the Pokemon gene, the distance between them was reduced to 254 and 106 bp. Our results showed that synergistic inhibition via the interaction between the NEG-U and NEG-D elements was almost abolished when the distance between the two elements was reduced, suggesting that synergistic inhibition also requires the regulatory ele- ments to be separated by a certain distance. It is likely that the appropriate distance facilitates DNA looping structure formation and is the threshold distance for the interaction between the NEG-U and NEG-D ele- ments. Deviation from the appropriate distance pre- vented the corresponding cis–trans complexes from acting synergistically with DNA looping to activate or suppress transcription. In conclusion, our studies are the first to elucidate the mechanism of the Pokemon gene transcription reg- ulation. Future studies will focus on the identification of proteins that can specifically bind to the NEG-U, NEG-D, and POS-D elements; this will provide a bet- ter understanding of the mechanisms of the Pokemon gene regulation. Experimental procedures Cells and cell culture Human lung carcinoma A549 cells were grown in Ham’s F12K medium containing 10% fetal bovine serum (Invitro- gen Corp., Carlsbad, CA, USA), human prostate carci- noma DU145 cells and human tumor of cervix uteri HeLa cells were grown in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Invitrogen), human hepatocyte carcinoma HepG2 cells and human acute T-cell leukemia cells were maintained in RPMI-1640 medium con- taining 10% fetal bovine serum (Invitrogen). All these cells were incubated in a humidified 5% CO 2 incubator at 37 °C. Creation of deletion constructs of the upstream region of the Pokemon gene The 2204-bp upstream region of the Pokemon gene, which spans the region )2220 to )17, was amplified by PCR from human blood genomic DNA by using the primers YU and YD (Table 1). The PCR products were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced; then, the 2204-bp promoter fragment was cloned into the BglII ⁄ HindIII sites of the pGL-3 basic vec- tor (Promega), and the resultant construct was designated as F-2220. The 5¢-deletion constructs with their endpoints Y. Yang et al. Analysis of upstream region of the Pokemon gene FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal compilation ª 2008 FEBS 1869 [...]... verify their fidelity, and they were termed WU-F, WU-I, MU-F, MU-I, WD-F, WD-I, MD-F and MD-I In order to determine the impact of the length Analysis of upstream region of the Pokemon gene between the NEG-U and NEG-D elements on the regulation of the Pokemon promoter, we inserted the NEG-U element into the KpnI ⁄ XhoI sites of F-381 and F-233 and sequenced them The resultant plasmids were termed D-254 and. .. CGGAACGCTGCTTCTCAAGGG-3¢; and M-D antisense primer, 5¢-CCCTTGAGAAGCAGCGTTCCGAAAGA AGACCCCCAGCCTCACATTCCCA-3¢ Mutated sites are underlined Construction of other reporter plasmids To determine the effect of the NEG-U and NEG-D elements on the activity of the SV40 promoter, we cloned these elements and their mutants into the KpnI ⁄ XhoI sites of the pGL3-control (Promega) in both the normal and reverse orientations The resultant... luciferase assay was performed using the dual luciferase assay kit (Promega) For control experiments, the MPOS-D, MNEG-U and MNEG-D double oligonucleotides were used as described in EMSAs To further determine the cooperation between the NEG-U and NEG-D elements, 2 lg of the NEG-U decoy, 2 lg of the NEG-D decoy, and a combination of 1 lg of each of the decoys were used, and the above-mentioned methods followed... technique assay To further determine whether the POS-D, NEG-U and NEG-D elements play important roles in regulation of the Pokemon promoter, the DNA decoy technique was employed Briefly, 300 ng of reporter plasmid, 30 ng of the pRL-TK vector, 0–2 lg of the double POS-D, NEG-U or NEG-D decoy oligonucleotides were cotransfected into A549 and DU145 cells using the lipofection method After 24 h of transfection,... sites and cloned into the pGL-3 basic vector The resultant constructs were mainly termed according to the nucleotide location of the 5¢-end of the forward primer used in the PCR reaction, e.g., F-2116, F-1712, F-837 and F-71 All the vector constructs were confirmed by DNA sequence analysis Because the ApaI site is located in the region between )88 and )83 of the Pokemon promoter, all 3¢-deletion constructs... in 24-well plates, and triplicate wells were set up for each group After the cells reached  90% confluence, they were transfected with of 30 ng of the pRL-TK vector and 300 ng of the pGL3 vectors containing different lengths of the Pokemon promoter fragment to each well by using LipofectamineTM2000 (Invitrogen) After 48 h of transfection, cells were harvested and lysed in 200 lL of reporter lysis buffer... underlined Site-directed mutagenesis analysis In order to further study the function of the NEG-U and NEG-D elements in the Pokemon promoter, base mutations were performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA) Three mutant constructs, MF-233 (the POS-D element was mutated in F-233), M-U (the NEG-U element was mutated in F-560) and M-D (the NEG-D element was mutated... 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Results Cloning of the 5¢-upstream region of the Pokemon gene To identify the regulatory sequences that control expression of the Pokemon gene, a 2204-bp section of the. of the Pokemon gene. Computer analysis of putative transcription factor-binding sites For a rough understanding of the regulation of the Pokemon gene, the 2204-bp section of its 5¢-upstream region. putative cis-elements in the 5¢-upstream region of the Pokemon gene. Analysis of upstream region of the Pokemon gene Y. Yang et al. 1862 FEBS Journal 275 (2008) 1860–1873 ª 2008 The Authors Journal