The 5V-Upstream Region of Human Programmed Cell Death 5 Gene Contains a Highly Active TATA-Less Promoter That Is Up-Regulated by Etoposide

http://www.paper.edu.cn Gene 329 (2004) 39–49 www.elsevier.com/locate/gene

The 5V-upstream region of human programmed cell death 5 gene contains a highly active TATA-less promoter that is up-regulated by etoposide

Mingxu Xu*, Ning Cheng, Liming Gui, Mouyi Lai, Ying Wang, Donglan Xia, Min Rui, Yingmei Zhang, Dalong Ma

Laboratory of Medical Immunology, School of Basic Medical Sciences, Peking University Center for Human Disease Genomics, 38 Xueyuan Road, Beijing 100083, China Received 9 June 2003; received in revised form 3 December 2003; accepted 23 December 2003

Received by R. Di Lauro

Abstract

The PDCD5 (programmed cell death 5), a novel apoptosis related gene, is functionally associated with cell apoptosis, exhibits a ubiquitous expression pattern and is up-regulated in some types of tumor cells undergoing apoptosis. To study the transcriptional regulation of the PDCD5 gene, we have cloned 1.1 kb of its 5V-upstream region. The DNA sequencing analysis revealed a major transcriptional start site at 72 base pairs in front of the ATG translational start codon. The upstream of the transcriptional start site lacks a canonical TATA box and CAAT box. Transient transfection and luciferase assay demonstrate that this region presents extremely strong promoter activity. The 5V- deleted sequences fused to a luciferase reporter gene demonstrated that the À 555/ À 383 region from the transcription start site is crucial for transcriptional regulation, and the luciferase reporter gene’s expression significantly increased in the early stage of cell apoptosis induced by etoposide. These results imply that the PDCD5 gene may be a target gene under the control of some important apoptosis-related transcriptional factors during the cell apoptosis. D 2004 Elsevier B.V. All rights reserved.

Keywords: PDCD5;5V-Upstream region; Etoposide; Apoptosis

1. Introduction (Yoshida et al., 2001), the mouse nuclear orphan receptor TR2-11 gene (Lee and Wei, 2000), the mouse orphan Apoptosis is a vital cellular process during tissue remod- nuclear receptor TEC gene (Maltais and Labelle, 2000), eling and differentiation throughout the life of an organism. and the human Fas gene (Cheng et al., 1995; Behrmann et It can be triggered by a variety of physiological and al., 1994). pathological stimuli, including radiation, drugs, growth In addition, a number of transcription factors involved in factor deprivation, hormones and stress (Earnshaw et al., the regulation of these genes expression have been identi- 1999; Meier et al., 2000; Yuan and Yankner, 2000). Many fied. The Brn-3a transcription factor stimulates expression gene products have been found to participate in the regula- of the anti-apoptotic Bcl-2 and Bcl-XL, protects neuronal tion of the apoptosis process, and the promoter regions of cells from apoptosis (Sugars et al., 2001); the E2F1 tran- some apoptosis-related genes have been cloned and charac- scription factor can control cell proliferation and apoptosis terized, such as the promoter regions of the rat caspase-3 (Lin et al., 2001); NF-nB enhances Bcl-XL expression via gene (Liu et al., 2002), the rat caspase-9 gene (Nishiyama et highly selective interactions with the Bcl-x promoter (Glas- al., 2001), the human death receptor 5/TRAIL-R2 gene gow et al., 2001); and NFAT, AP-1 and Sp1 are essential for regulation of the expression of TNF family members (Wang et al., 2000). Abbreviations: PDCD5, programmed cell death 5; kb, kilobase(s); RT, reverse transcription; PMSF, phenylmethylsulfonyl fluoride. Using the cDNA-representative differences analysis * Corresponding author. Tel./fax: +86-10-82801149. (cDNA-RDA) approach, we have identified a novel apopto- E-mail address: [email protected] (M. Xu). sis-related gene—PDCD5 (programmed cell death 5), from

40 M. Xu et al. / Gene 329 (2004) 39–49

TF-1 cells undergoing apoptosis (formerly named TF-1 cell subcloned into pGEM-T Easy vector (Promega) to obtain apoptosis related gene-19, TFAR19) (Liu et al., 1999). plasmid pGEM-PDCD5 and then subjected to DNA sequenc- PDCD5 protein is conservative in the process of evolution ing by the dideoxynucleotide chain-termination method us- and has significant homology to the corresponding proteins inganABIautomatedDNAsequencer(3100Genetic of species from yeast to mouse (Liu et al., 1999; Song et al., Analyzer). The nucleotide sequences were determined for 1999).ThePDCD5 mRNA was widely expressed in a both DNA strands using vector-specific T7 and SP6 primers. variety of normal tissues and the expression of its mRNA in fetal tissues was significantly lower than that in adult 2.2. Determination of the transcription start site tissues. Both of the levels of PDCD5 mRNA and protein in TF-1 cells were increased in the process of apoptosis. To obtain full-length PDCD5 cDNA sequence, a Data- Overexpressed PDCD5 or recombinant PDCD5 protein base search was performed at the National Center for could accelerate apoptosis of some tumor cells including Biotechnology Information (NCBI; http://www.ncbi.nlm. HeLa, TF-1, HL60, MCG-803, and MCF-7 cells, etc. (Liu et nhi.gov/) and Database of transcriptional Start Sites al., 1999; Zhang et al., 2000). Using specific monoclonal (DBTSS; http://elmo.ims.u-tokyo.ac.jp/dbtss) (Suzuki et antibodies against PDCD5, we have found that PDCD5 al., 2002). Reverse transcription PCR (RT-PCR) was used protein can rapidly translocate to the nucleus in the cells to validate the results of the Database sequence searching. undergoing apoptosis and the accumulation of PDCD5 Total RNA was isolated from HeLa cells using TriZOL (Life protein in the nucleus precedes the chromosome DNA Technologies) following the manufacturer’s recommenda- fragmentation and phosphatidylserine (PS) externalization tion. Reverse transcriptional reactions were performed using (Chen et al., 2001), indicating PDCD5 protein may be a 2 Ag of total RNA, MMLV RT (Gibco) and the antisense vital molecule for apoptosis. However, the transcriptional primer (Xu4, described below). The cDNA was amplified regulation of the PDCD5 gene remains unclear. To investi- using primer pairs as follows: Sense primer (Xu25) and gate the regulation of the PDCD5 gene at the transcriptional antisense primer (Xu4); Sense primer (Xu26) and antisense level, we isolated and characterized the 5V-upstream region primer (Xu4). The genomic 1.1-kb pair PCR fragment was of this gene. The results suggest that the 1.1 kb of the 5V- used as a positive control. Xu4: 5V-CAAGCTCCTCGTC- upstream region of this gene contains several potential CGCCATG-3V ( À 1/ + 19 from ATG start codon) Xu25: 5V- regulatory elements and can induce the expression of the CCCCGCGAGCGCCTGCGC-3V ( À 90/ À 73 from ATG luciferase reporter gene in HeLa cells. By deletion muta- start codon) Xu26: 5V-AGTGGTCAAGGCCGCGCTCG- genesis analysis, it was shown that the À 555-bp upstream 3V ( À 72/ À 53 from ATG start codon). fragment of the PDCD5 gene is sufficient to drive the transcription of the luciferase reporter gene and that the 2.3. Construction of luciferase reporter vector core promoter resides in the sequence spanning À 555 to À 383 bp from the transcription start site. Moreover, the A 1.1-kb genomic fragment of the 5V-upstream region of luciferase reporter gene’s expression is significantly in- the PDCD5 gene was generated by digesting the plasmid creased at the early stage of cell apoptosis induced by pGEM-PDCD5 with EcoRI and subcloned into the SmaI etoposide. site of the promoter-less luciferase reporter gene vector pGL3-enhancer (Promega) in both the forward and reverse orientations to obtain plasmids pGL3-TFFw or pGL3-TFRv. 2. Materials and methods Sequencing was performed on both DNA strands of the reporter plasmids. The sequencing data were analyzed and 2.1. Cloning of the 5V-upstream region of the PDCD5 gene compared with the human genomic sequence using the BLAST from NCBI. Human genomic DNA was isolated from 293T cell lines By using a reverse primer (5V-CTAGCTAGCAA- according to the phenol/chloroform extraction protocol. GCTCCTCGTCCGCCATG-3V, À 1/ + 19 from ATG start Based on the published sequence for human PDCD5 ge- codon) and two different forward primers (Fw1: 5V- nomic DNA (GenBank accession No. AC008474), we CTAGCTAGCTTTAGGACATTTC-3V, À 455/440 from designed a sense primer (5V-CTTGAGCTCAGGAGATA- ATG start codon; Fw2: 5V-CTAGCTAGCCCAAGTCCC- GAG-3V, À 1085/ À 1066 from ATG start codon) and an TGCAC-3V, À 273/ À 257 from ATG start codon), two antisense primer (5V-CAAGCTCCTCGTCCGCCATG-3V, progressive deletion sequences from ATG start codon À 1/ + 19 from ATG start codon). The 5V-upstream region À 455/ + 19 and À 273/ + 19 were amplified using the of the PDCD5 gene was amplified by PCR (GenAmpRPCR plasmid pGEM-PDCD5 as template and subcloned into System 9700, PE Applied Bio Systems) using the DNA pGL3-enhancer, designated pGL3-TF5 and pGL3-TF6, re- amplification kit (TaKaRa) and LA Taq polymerase follow- spectively. All the specific complementary sequences in the ing the manufacturer’s protocol. The PCR profile was 4 min primers were preceded by an arbitrary sequence including a at 94 jC for 1 cycle, 20 s at 96 jC and 2 min at 68 jC for 35 NheI restriction site. The constructs were verified for cycles followed by 7 min at 72 jC. The PCR product was orientation by sequencing. 中国科技论文在线 http://www.paper.edu.cn

M. Xu et al. / Gene 329 (2004) 39–49 41

Digestion of primary construct pGL3-TFFw with KpnI containing 25 mM HEPES, pH 7.5, 0.1%CHAPS and 10 and either PvuII, PstIorSpeI and subsequent self-ligation mM dithiothreitol with the fluorogenic substrate z-DEVD- large fragment generated another three deletion constructs, AMC (Pharmingen). To measure the signal, we used the designated pGL3-KPv, pGL3-KPs and pGL3-KSp encom- POLARstar galaxy spectrometer (BMG Labtechnologies) passing the sequences from the ATG start codon À 871/ using an excitation filter of 380 nm and emission filter of + 19, À 714/ + 19 and À 627/ + 19, respectively. 460 nm. Results were reported as the percentage of control over 90 min (T90 to T0). All samples were prepared in 2.4. Cell culture, transfections and luciferase assay triplicate.

HeLa and 293T cells were cultured in Dulbecco’s mod- 2.7. Detection of PS externalization ified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (FCS) at 37 jC in a 5% CO2 atmosphere. HeLa cells were treated with etoposide, then were For the transient transfection assay, cells were seeded into washed and re-suspended in binding buffer containing 24-well plates 1 day before transfection. The medium was FITC-conjugated annexin V (25 Ag/ml) for 15 min prior renewed 2 h before transfection. Cells were transfected by to analysis. Cells were collected on a FACScan flow the calcium phosphate precipitation method. Transfection cytometer equipped with a 488-nm argon laser and analyzed reactions contained total 1.2 Ag of plasmid DNA/well, using the CellQuest software (Becton Dickinson). consisting of 1 Ag of reporter plasmid and 0.2 Agof Additionally, treated cells were also incubated with the pGFP-N1 (Clontech) to correct for transfection efficiency. R-PE-conjugated Annexin V (0.5 Ag/ml final concentra- DNA precipitates were incubated with cell cultures over- tion), followed by fluorescence microscope analysis. night. luciferase activity was assayed 24 h after transfection. Cells were washed with phosphate-buffered saline and lysed 2.8. Immunofluorescence analysis with 50 Al of freshly diluted reporter lysis buffer (Promega). After centrifugation, 10 Al of supernatant was added to 50 The cells stained with the R-PE-conjugated Annexin V Al of the luciferase assay substrate (Promega) and the were washed in PBS twice, fixed in freshly prepared 2% luminescence of the samples were read immediately with paraformaldehyde (containing 0.1% Triton-X 100) for 30 a POLARstar galaxy spectrometer (BMG Labtechnologies), min at room temperature, pre-incubated in 2% fetal bovine in which light production (relative light units) was measured serum (FBS) for 1 h and incubated with FITC labeled anti- for 10 s. The results were normalized with equivalent PDCD5 monoclonal antibody (FITC-3A3) for 1 h. Follow- quantities of GFP proteins. The experiment was repeated ing antibody staining, cells were observed under the fluo- at least three times. rescence microscope.

2.5. Apoptosis induction and luciferase assay 3. Results HeLa cells were used for induction of apoptosis. The methods of cell culture and transfection have been described 3.1. Cloning of the 5V-upstream region of PDCD5 gene above. Briefly, HeLa cells were cultured in DMEM containing 10% FCS. The cells were seeded into 24-well plates Using the sense and antisense primer, the 1.1-kb frag- the day before transfection. The cells were transfected with ment was amplified from human genomic DNA. Upon pGL3-TFFw ( À 1085/ + 19 from ATG start codon), pGL3- sequencing with T7 and SP6 primers, it exhibited the KPs ( À 714/ + 19), pGL3-TF5 ( À 455/ + 19), and pGL3- presence of 43 bp of the first exon (original clone) and promoter (including a SV40 promoter) constructs by the the 1061-bp 5V flanking genomic region of PDCD5 gene. calcium phosphate method. After 24 h, etoposide (120 Ag/ This result indicated that the cloned fragment was physically ml) was added to the medium for induction of apoptosis. linked to exon 1 of PDCD5 gene and contained the 5V- Assay of luciferase activity and detection of apoptosis were upstream region. Hence, this fragment was used for further carried out 2, 4, 6 and 8 h after the addition of etoposide. studies. The method of luciferase assay has been described above. 3.2. Structure of human PDCD5 gene 2.6. Measurement of caspase activity The comparison of the PDCD5 genomic DNA and Drug-treated HeLa cells were washed twice with pre-cold cDNA suggested that the human PDCD5 gene consists of phosphate-buffered saline and harvested in lysis buffer (10 six exons spanning approximately 6 kb (Fig. 1). The exons mM Tris, pH 7.5, 10 mM Na2HPO4/NaH2PO4, 130 mM of the PDCD5 gene varied in size from 36 to 168 bp, and NaCl, 1% Triton-X100, 1 mM PMSF). The cell lysates were the introns range in size from 323 to 2718 bp. All of the then clarified by centrifugation at 4 jC. Cell lysates con- exon/intron boundary sequences are consistent with the GT/ taining 15 Ag of protein were incubated at 37 jC in a buffer AG rule (Table 1). 中国科技论文在线 http://www.paper.edu.cn

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Fig. 1. Schematic representation of the PDCD5 gene and cDNA structure. The boxes show the exons with their relative size and the positions in the PDCD5 gene. Numerals above the boxes indicate exon numbers.

3.3. Mapping of the transcription start site of the PDCD5 for the 300 bases proximal to the ATG start codon (data not promoter shown). In addition, this region contains potential binding sites for several transcription factors, such as Sp1, ETF, For identification of PDCD5 gene transcription start GATA1 and NF-IL6, et al. The presence of these multiple site(s), a Database search of the NCBI and DBTSS were transcription factor binding sites in the region further performed. From DBTSS of the Database, we found a full- suggested that the 1.1-kb fragment contains a functional length cDNA sequence of PDCD5, which was determined promoter. by an oligo capping method and elongated 48 bp at 5V- upstream region compared with the original clone (Fig. 2A). 3.5. Promoter activity of the cloned fragment The location of the transcription start site was confirmed by RT-PCR analysis using primers flanking the start site (Fig. To determine whether the genomic DNA fragment con- 2B). Primer pair 1 (Xu26, sequence + 1 to + 20 bp from tains a functional promoter, the 1.1-kb fragment of the 5V- transcription start site and Xu4) generated a PCR product on upstream region DNA was cloned into a promoter-less the RNA template, whereas primer pair 2 (Xu25, sequence luciferase reporter plasmid pGL3-enhancer upstream of the À 1to À 18 bp from transcription start site and Xu4) did luciferase gene in both orientations (pGL3-TFFw and not. As a control, PCR was carried out with a genomic DNA pGL3-TFRv). These recombinant plasmids were transfected template using both primer pairs (Fig. 2B). Using a combi- into HeLa cells and cell lysates were assayed for luciferase nation of Database searching and the results of RT-PCR, we activity 24-h post-transfection. The forward construct inferred that 72-bp 5V upstream of ATG translation start pGL3-TFFw exhibited extremely strong promoter activity codon should be a transcription start site ( + 1). (Fig. 4A). No promoter activity was observed in the transfection of the reverse construct pGL3-TFRv. In addi- 3.4. Sequence analysis of the 5V-upstream region tion, the same experiment was performed in 293T cells and similar results were observed (data not shown). To characterize the potential regulatory sequences in- In order to localize the core promoter in PDCD5 gene volved in PDCD5 gene expression, the 5V-upstream region and to assess the roles of different trans-acting factor of the PDCD5 gene was sequenced and analyzed. Fig. 3 binding motifs in regulation of basal PDCD5 gene expres- shows the 1.1-kb sequence of the 5V-upstream region from sion, sequential unidirectional deletion analysis of the 1.1- the ATG start codon. Sequence analysis revealed that the kb fragment from the 5V-upstream region was carried out PDCD5 gene contains neither a canonical TATA-like ele- (Fig. 4B). The deletion constructs, pGL3-KPv and pGL3- ment nor a CAAT box in close proximity to the transcription KPs ( À 799/ + 19 and À 642/ + 19 from transcription start start site. However, the 5V-upstream region has a high GC site) exhibited almost the same luciferase activity compared content. The overall GC content is 54.5%, while it is 76.9% to the construct containing the entire 1.1-kb fragment

Table 1 Nucleotide sequences of the exon/intron boundaries in PDCD5 genea No. Exon size (bp) 5V Splice donor Intron size (bp) 5V Splice donor 190 CAAACACGGG gtgagcgcat 867 ttttttccag GATCCTGGTG 238 CAAAGCACAG gtatgggctg 2718 ttcgttgtag GGAAGCAGAA 362 CGGGCCAGGT gtaagcatct 804 ttctttttag TAAGTAACTT 492 AAGTGAGAAG gtaagcttag 950 gttctcctag GTATCAGAAC 572 AACAGTGAAA gtaagtgtcc 323 ttcctcccag TTCAACAGAA 6 168 TTATGCAAAA a The nucleotide sequence of each exon/intron boundary and the sizes of the exon and the intron are shown. Exon sequences are in capital letters; intron sequences are in lower case letters. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 2. Mapping of the transcription start site of the PDCD5 promoter. (A) Sequence of exon 1 of PDCD5 cDNA. Boldface show original clone of partial exon 1 and italics indicated part of elongation. (B) The result of RT-PCR. PCR analysis was performed with 1 Ag of total HeLa cells RNA or genomic DNA. Lanes 1 and 2 show the DNA Marker, DL2000 ladder. Lanes 2 and 3 show the PCR for genomic DNA; lanes 4 and 5 show the PCR for RNA.

Fig. 3. Nucleotide sequence of the 5V-upstream region of the PDCD5 gene. The region includes several kinds of potential cis-acting regulatory elements including multiple SP1-binding sites and ETF core sequences (double underlined), E2A binding sites and E Boxes (Boxes) and several other elements (underline). + 1 shows the position of the transcription start site. The first exon sequence is in italics. The translation start codon is shown in boldface. 中国科技论文在线 http://www.paper.edu.cn

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Fig. 4. Transcription activity of the PDCD5 gene 5V-upstream region. (A) Promoter activity of the PDCD5 gene 5V-upstream region. The left and right panels show the structures of the constructs and the results of luciferase assay, respectively. (B) Deletion analysis of the PDCD5 gene 5V-upstream region. The left panel shows the 5V step-wise deletion constructs used to test the functional activity of the PDCD5 gene 5V-upstream region in transient transfection assays. Numbers to the left of each construct indicate the 5V end of the upstream region fragment relative to the ATG start codon of the PDCD5 gene. The right panel shows the results of the luciferase assays of various 5V deletion constructs. The various deletion constructs were transfected into HeLa cells. The luciferase activities, normalized with GFP activities, were expressed as a percentage of the negative control (i.e., full-length reverse construct pGL3-TFRv). Each construct was transfected three times and assayed in duplicate.

(pGL3-TFFw, À 1013/ + 19 from transcription start site). caspase-3 activity measured by fluorogencic substrate z- Upon further deletion, a noticeable but nonsignificant de- DEVD-AMC. There was a 28% increase in caspase-3 crease was observed with the construct pGL3-KSp ( À 555/ activity 2 h after the addition of etoposide and a 1.6-fold + 19 from transcription start site) compared to the pGL3- increase after 8 h compared to the control group (Fig. 5A). TFFw construct. Further successive 5V-deletion of the pro- The detection of PS externalization with Annexin-V-FITC moter sequence to À 383 (pGL3-TF5) markedly decreased indicated similar results to the measurement of caspase-3 promoter activity in HeLa cell lines, indicating that some activity (Fig. 5B). Additionally, to prove that PDCD5 gene positive cis-elements are located in this region ( À 555/ activation was directly linked to etoposide-induced apo- À 383). Further stepwise removal of sequence spanning ptosis in this experiment system, apoptotic cells were between À 201 and + 91 produced a low promoter activity detected on the basis of the PS externalization using R- compared to the À 1013/ + 91 reverse construct pGL3- PE-conjugated Annexin V, and expression level of endog- TFRv (negative control). enous PDCD5 gene was detected using FITC-3A3 simul- taneously (Fig. 5C). The results show that higher 3.6. Up-regulation of PDCD5 gene promoter by etoposide percentages of apoptotic cells (over the 70%) were observed 2 h after the addition of etoposide. In parallel, the To assess the effects of PDCD5 gene promoter region level of PDCD5 expression was markedly up-regulated in on the regulation of PDCD5 gene expression during cell most of the apoptotic cells. apoptosis, we transfected a full-length construct (pGL3- Assay of luciferase activity was carried out 2, 4, 6 and 8 h TFFw) or a promoter control plasmid pGL3-promoter after addition of etoposide. The results of the luciferase (only containing SV40 promoter) into HeLa cells. The assay are shown in Fig. 5D. The luciferase activity of the chemotherapy drug etoposide was applied to induce apo- report gene construct (pGL3-TFFw) was much higher than ptosis after 24 h of transfection (DMSO was added in that of the DMSO at 2, 4 and 6 h after treatment of control). Two methods to detect apoptosis were carried out etoposide, while no significant difference was observed at at 0, 2 and 8 h after addition of etoposide. Results of these 8 h. These results may suggest that the PDCD5 gene 5V- assays are shown in Fig. 5A and B. After etoposide upstream region can promote the expression of downstream treatment of HeLa cells, we observed an increase in gene significantly during the early phase of apoptosis. This 中国科技论文在线 http://www.paper.edu.cn

M. Xu et al. / Gene 329 (2004) 39–49 45

Fig. 5. Time course of apoptosis and the promoter activity of PDCD5 gene in HeLa cells after treatment with etoposide. (A) The results of measurement of caspase-3 activity. Fifteen-microgram cell lysates of protein were incubated at 37 jC in a buffer containing fluorogenic substrate z-DEVD-AMC and caspase-3

activity measured by POLARstar galaxy spectrometer. Results are shown as a percentage of the control over 90 min (T90 to T0). (B) The results of detection of PS externalization. HeLa cells were treated with etoposide and PS externalization was determined by flow cytometry. Similar experiments were done in duplicate. (C) The results of the fluorescence microscope analysis. HeLa cells were treated with etoposide and incubated with the R-PE-conjugated Annexin V, followed by fluorescence microscope analysis (a: untreated group, b: treated group). Subsequently, the cells incubated with FITC-3A3 and were observed under the fluorescence microscope (c: untreated group, d: treated group). (D) The promoter activity of PDCD5 gene in HeLa cells after stimulation with etoposide. HeLa cells transfected with the plasmid pGL3-TFFw ( À 1085/ + 19) and pGL3-SV40 promoter were incubated with etoposide and DMSO for various time periods (left panel). Transfection efficiency was normalized by GFP activity. The results of luciferase assay were shown in right panel. 中国科技论文在线 http://www.paper.edu.cn

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is correlated with our earlier studies that expression of To identify the core region in the 5V-upstream sequence PDCD5 gene is up-regulated at the same phase (Liu et al., of the PDCD5 gene that is responsible for apoptosis, we 1999). Furthermore, the luciferase activity of the promoter transfected full-length construct (pGL3-TFFw) and two control plasmid indicates no significant difference compared deletion constructs (pGL3-KPs and pGL3-TF5) into HeLa with that of DMSO within various time intervals after cell lines (Fig. 6). After transfection of the deletion construct apoptosis induction. Together above observation, increase pGL3-KPs, the results were similar to those of the full- in luciferase activity induced by epotoside is specific for length construct. However, after transfection of the deletion PDCD5 promoter. construct pGL3-TF5, the expression level of the report gene

Fig. 6. Deletion analysis of the PDCD5 gene 5V-upstream region in HeLa cells after stimulation with etoposide. (A) The 5V deletion constructs used to test are shown. (B) The results of luciferase assays of various 5V deletion constructs after stimulation with etoposide. HeLa cells were transiently transfected with the indicated luciferase constructs and incubated with 120 Ag/ml etoposide for various time periods. The luciferase assay was carried out as described under the experimental procedures. Transfection efficiency was normalized by GFP activity. The luciferase activity ratio of the treatment to the control is shown. The results of luciferase assay carried out 2, 4, 6 and 8 h after stimulation with etoposide are shown in a, b, c and d, respectively. 中国科技论文在线 http://www.paper.edu.cn

M. Xu et al. / Gene 329 (2004) 39–49 47

exhibits no significant difference compared with that of sites for LBP-1, E2A, GATA-1 and GCF within this region DMSO within various time intervals after apoptosis induc- and the subsequent decrease of promoter activity associated tion. These results suggest that the À 642/ À 383 sequence with the deletion of this region suggest that this 5V-upstream of the PDCD5 gene promoter region is correlated with portion of PDCD5 gene promoter is important for transcrip- apoptosis. Sequencing analysis of this region shows that tional activity in HeLa cells. Further deletion construct several potential cis-elements present this sequence, such as pGL3-TF6 ( À 201/ + 91) produced an appreciable drop in E box, E2A and GATA-1, etc. (Fig. 3). the promoter activity as compared to the pGL3-TF5 construct. Several motifs like SP1, AP2, ETF, GATA-1, etc. exist in this region. Thus, we can infer that the sequence 4. Discussion between À 201 and + 91 may be functional when sequence between À 555 and À 383 is present. The PDCD5 gene was cloned from TF-1 cells undergo- A variety of experimental results have suggested that the ing apoptosis in our laboratory in 1999. Early studies chemotherapy drug etoposide could induce apoptosis in indicate that the expression level of PDCD5 is obviously susceptible cell lines, such as MCF7 cells (Benjamin et up-regulated in the process of apoptosis. Recombinant al., 1998), U-937 cells (Martinsson et al., 2001), HL-60 cells PDCD5 protein or overexpression of PDCD5 could accel- (Martins et al., 1997) and HeLa cells (Mizukami et al., erate apoptosis of some types of tumor cells (Liu et al., 1999; Negri et al., 1993; Bernardi et al., 1995). In addition, 1999). In the process of the cells undergoing apoptosis, caspase-3 was activated in the process of apoptosis induced PDCD5 protein rapidly translocates to the nucleus, and by different methods in a variety of cell types. These accumulates in the nucleus proceeding the chromosome included cytotoxic T cells treated with Fas ligand (Schlegel DNA fragmentation and phosphatidylserine (PS) external- et al., 1996), human U-937 cells treated with 1-beta-D- ization (Chen et al., 2001). Those results showed that the arabinofuranosyl-cytosine (Datta et al., 1996), MCF7 cells PDCD5 gene was functionally linked to apoptosis. This and HeLa cell treated with etoposide (Benjamin et al., 1998; raises the question regarding the transcriptional regulation Mizukami et al., 1999). We used etoposide to induce of the PDCD5 gene expression. In this study, the human apoptosis of HeLa cells and the results indicated that the PDCD5 gene 5V-upstream region has been isolated and typical features of apoptosis were visible in HeLa cells by characterized. the flow cytometry, the measurement of caspase-3 activity Sequence analysis of the 1.1-kb 5V-upstream region and the fluorescence microscope analysis (Fig. 5A–C).In revealed that the PDCD5 gene has a TATA-less promoter parallel, the PDCD5 expression was observably increased in with a high GC content, which is similar to some other the apoptotic cells. These results suggested that PDCD5 apoptosis-associated gene. For example, the 5V-upstream activation was directly linked to apoptosis. promoter region of murine TRAF1 lacks TATA-like and The luciferase assay showed that the promoter activity of CAAT-like sites but contains GC-rich sequences (Dunn et the 1.1-kb region was up-regulated by etoposide and re- al., 1999); the promoter region of the murine p75 TNF markably decreased when the region between À 642 and receptor (TNF-R) is devoid of TATA box and has the À 383 nt was deleted. This region sequence contains one characteristics of a house keeping gene since it contains E2A binding site and two E boxes (CANNTG) (Fig. 2). The multiple SP1 binding sites (Kissonerghis et al., 1999); and E2A binding site and E boxes are located in the enhancers analysis of the 5V flanking region of the human survivin and promoters of genes regulated by the bHLH (basic helix- gene revealed the presence of a TATA-less promoter con- loop-helix) transcription factors. E2A, one of the bHLH taining a canonical CpG island (Li and Altieri, 1999). family members, also interacts with E box. The bHLH Luciferase activity assays indicated that the reporter gene proteins are ubiquitously expressed transcription factors construct containing the entire 1.1-kb 5V-upstream region, and play a pivotal role in the regulation of cell growth i.e. pGL3-TFFw, exhibited extremely strong promoter ac- and differentiation. Several lines of evidence also indicate tivity, suggesting that the cloned 5V-upstream region of the that bHLH transcription factors play important roles during PDCD5 gene contains a functional promoter. cell apoptosis. E2A and HEB, two basic helix-loop-helix The deletion constructs, pGL3-KPv, pGL3-KPs and transcription factors, can negatively regulate pre-TCR and pGL3-KSp (deletion of 214, 371 and 458 bp, respectively, TCR signaling, their removal causing hyperactivation and from the 5V end of the PDCD5 gene upstream region) apoptosis of thymocytes (Kim et al., 2002). The expression almost exhibited the same level of luciferase activity com- of E2A-HLF chimeric protein, encoding a chimeric tran- pared to the construct containing the entire 1.1-kb region scription factor in which the trans-activating domains of (pGL3-TFFw). These results suggest that the cis-element E2A are fused to the DNA-binding and dimerization present between À 1013 and À 555maynotplayan domains of hepatic leukemic factor (HLF), induced T-cell important role in the PDCD5 gene promoter activity of apoptosis, B-cell maturation arrest, and development of HeLa cells. However, deletion construct pGL3-TF5 (À 383/ acute lymphoblastic leukemia (Honda et al., 1999); The + 19) showed a marked decrease promoter activity as entopic expression of a bHLH gene Math1 is toxic to compared to pGL3-TFFw. The presence of putative binding neurons and leads to apoptosis (Isaka et al., 1999); the 中国科技论文在线 http://www.paper.edu.cn

48 M. Xu et al. / Gene 329 (2004) 39–49

chimeric homeodomain protein E2A-PBX1 paradoxically Glasgow, J.N., Qiu, J., Rassin, D., Grafe, M., Wood, T., Perez-Pol, J.R., induces both proliferation and apoptosis in lymphoid cells 2001. Transcriptional regulation of the BCL-X gene by NF-kappaB is an element of hypoxic responses in the rat brain. Neurochem. Res. 26, (Dedera et al., 1993; Smith et al., 1997). 647–659. From the above findings, we can conclude that the À 642/ Honda, H., Inaba, T., Suzuki, T., Oda, H., Ebihara, Y., Tsuiji, K., À 383-bp sequence of the PDCD5 gene 5V-upstream region Nakahata, T., Ishikawa, T., Yazaki, Y., Hirai, H., 1999. Expression is crucial for transcriptional regulation of PDCD5 gene of E2A-HLF chimeric protein induced T-cell apoptosis, B-cell mat- expression during apoptosis in HeLa cells. Furthermore, we uration arrest, and development of acute lymphoblastic leukemia. Blood 93, 2780–2790. deduce that bHLH transcription factors binding E box may Isaka, F., Ishibashi, M., Taki, W., Hashimoto, N., Nakanishi, S., Kageyama, play a key role in inducing expression of PDCD5 during R., 1999. Ectopic expression of the bHLH gene Math1 disturbs neural apoptosis. development. Eur. J. Neurosci. 11, 2582–2588. In summary, the 5V-upstream region DNA of the human Kim, D., Xu, M., Nie, L., Peng, X.C., Jimi, E., Voll, R.E., Nguyen, T., PDCD5 gene was cloned and it was demonstrated that Ghosh, S., Sun, X.H., 2002. Helix-loop-helix proteins regulate pre-TCR and TCR signaling through modulation of Rel/NF-kappaB activities. extremely strong promoter activity existed in this region. Immunity 16, 9–21. Deletion studies indicated that the cis-element present Kissonerghis, M., Daly, G., Feldmann, M., Chernajovsky, Y., 1999. The between À 555/ À 383-bp sequence might play pivotal role murine p75 TNF receptor promoter region: DNA sequence and charac- in the PDCD5 gene promoter activity. In addition, the terization of a cis-acting silencer. Mol. Immunol. 36, 125–134. À 642/ À 383-bp sequence of 5V-upstream region was cru- Lee, C.H., Wei, L.N., 2000. Characterization of the mouse nuclear orphan receptor TR2-11 gene promoter and its potential role in retinoic acid- cial for transcriptional regulation of PDCD5 gene expres- induced P19 apoptosis. Biochem. Pharmacol. 60, 127–136. sion during apoptosis induced by etoposide. Li, F., Altieri, D.C., 1999. Transcriptional analysis of human survivin gene expression. Biochem. J. 344, 305–311. Lin, S.C., Skapek, S.X., Papermaster, D.S., Hankin, M., Lee, E.Y., 2001. Acknowledgements The proliferative and apoptotic activities of E2F1 in the mouse retina. Oncogene 20, 7073–7084. We thank Prof. Jiaping Tao for making available the Liu, H.T., Wang, Y.G., Zhang, Y.M., Song, Q.S., Di, C.H., Chen, G.H., Tang, J., Ma, D.L., 1999. TFAR19, a novel apoptosis-related gene Becton Dickinson FACScan flow cytometer used in this cloned from human leukemia cell line TF-1, could enhance apoptosis study. We are also grateful to Madam Daniel Calvin and Dr. of some tumor cells induced by growth factor withdrawal. Biochem. Zhengren Wei for the excellent assistance in the reading of Biophys. Res. Commun. 254, 203–210. the manuscript. Liu, W., Wang, G., Yakovlev, A.G., 2002. Identification and functional This work was supported by Grants from the National analysis of the rat caspase-3 gene promoter. J. Biol. Chem. 277, 8273–8278. Natural Sciences Foundation of China (30170871). Maltais, A., Labelle, Y., 2000. Structure and expression of the mouse gene encoding the orphan nuclear receptor TEC. DNA Cell Biol. 19, 121–130. References Martins, L.M., Kottke, T., Mesner, P.W., Basi, G.S., Sinha, S., Frigon Jr., N., Tatar, E., Tung, J.S., Bryant, K., Takahashi, A., Svingen, Behrmann, I., Walczak, H., Krammer, P.H., 1994. Structure of the human P.A., Madden, B.J., McCormick, D.J., Earnshaw, W.C., Kaufmann, APO-1 gene. Eur. J. Immunol. 24, 3057–3062. S.H., 1997. Activation of multiple interleukin-1beta converting en- Benjamin, C.W., Hiebsch, R.R., Jones, D.A., 1998. Caspase activation in zyme homologues in cytosol and nuclei of HL-60 cells during eto- MCF7 cells responding to etoposide treatment. Mol. Pharmacol. 53, poside-induced apoptosis. J. Biol. Chem. 272, 7421–7430. 446–450. Martinsson, P., Liminga, G., Nygren, P., Larsson, R., 2001. Characteristics Bernardi, R., Negri, C., Donzelli, M., Guano, F., Torti, M., Prosperi, E., of etoposide-induced apoptotic cell death in the U-937 human lympho- Scovassi, A.I., 1995. Activation of poly(ADP-ribose)polymerase in ap- ma cell line. Anticancer Drugs 12, 699–705. optotic human cells. Biochimie 77, 378–384. Meier, P., Finch, A., Evan, G., 2000. Apoptosis in development. Nature Chen, Y.Y., Sun, R.H., Han, W.L., Zhang, Y.M., Song, Q.S., Di, C.H., Ma, 407, 796–801. D.L., 2001. Nuclear translocation of PDCD5 (TFAR19): an early signal Mizukami, S., Kikuchi, K., Higuchi, T., Urano, Y., Mashima, T., Tsuruo, T., for apoptosis? FEBS Lett. 509, 191–196. Nagano, T., 1999. Imaging of caspase-3 activation in HeLa cells stim- Cheng, J., Liu, C., Koopman, W.J., Mountz, J.D., 1995. Characterization ulated with etoposide using a novel fluorescent probe. FEBS Lett. 453, of human Fas gene. Exon/intron organization and promoter region. 356–360. J. Immunol. 154, 1239–1245. Negri, C., Bernardi, R., Braghetti, A., Ricotti, G.C., Scovassi, A.I., 1993. Datta, R., Banach, D., Kojima, H., Talanian, R.V., Alnemri, E.S., Wong, The effect of the chemotherapeutic drug VP-16 on poly(ADP-ribosyla- W.W., Kufe, D.W., 1996. Activation of the CPP32 protease in apo- tion) in apoptotic HeLa cells. Carcinogenesis 14, 2559–2564. ptosis induced by 1-beta-D-arabinofuranosylcytosine and other DNA- Nishiyama, J., Yi, X., Venkatachalam, M.A., Dong, Z., 2001. cDNA clon- damaging agents. Blood 88, 1936–1943. ing and promoter analysis of rat caspase-9. Biochem. J. 60, 49–56. Dedera, D.A., Waller, E.K., LeBrun, D.P., Sen-Majumdar, A., Stevens, Schlegel, J., Peters, I., Orrenius, S., Miller, D.K., Thornberry, N.A., Yamin, M.E., Barsh, G.S., Cleary, M.L., 1993. Chimeric homeobox gene E2A- T.T., Nicholson, D.W., 1996. CPP32/apopain is a key interleukin 1 beta PBX1 induces proliferation, apoptosis, and malignant lymphomas in converting enzyme-like protease involved in Fas-mediated apoptosis. transgenic mice. Cell 74, 833–843. J. Biol. Chem. 271, 1841–1844. Dunn, I.F., Geha, R.S., Tsitsikov, E.N., 1999. Structure of the murine Smith, K.S., Jacobs, Y., Chang, C.P., Cleary, M.L., 1997. Chimeric onco- TRAF1 gene. Mol. Immunol. 36, 611–617. protein E2a-Pbx1 induces apoptosis of hematopoietic cells by a p53- Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian cas- independent mechanism that is suppressed by Bcl-2. Oncogene 14, pases: structure, activation, substrates, and functions during apoptosis. 2917–2926. Annu. Rev. Biochem. 68, 383–424. Song, Q.S., Han, W.L., Liu, H.T., Ma, D.L., 1999. The cDNA cloning and 中国科技论文在线 http://www.paper.edu.cn

M. Xu et al. / Gene 329 (2004) 39–49 49

sequencing of a novel mouse apoptosis related gene TFAR19. J. Beijing Yoshida, T., Maeda, A., Tani, N., Sakai, T., 2001. Promoter structure and Med. Univ. 31, 309–311. transcription initiation sites of the human death receptor 5/TRAIL-R2 Sugars, K.L., Budhram-Mahadeo, V., Packham, G., Latchman, D.S., 2001. gene. FEBS Lett. 507, 381–385. A minimal Bcl-x promoter is activated by Brn-3a and repressed by p53. Yuan, J., Yankner, B.A., 2000. Apoptosis in the nervous system. Nature Nucleic Acids Res. 29, 4530–4540. 407, 802–809. Suzuki, Y., Yamashita, R., Nakai, K., Sugano, S., 2002. DBTSS: DataBase Zhang, Y.M., Xu, X.Z., Liu, H.T., Song, Q.S., Ma, D.L., 2000. The apo- of human Transcriptional Start Sites and full-length cDNAs. Nucleic ptosis-accelerating effect of human recombinant TFAR19 protein on Acids Res. 30, 328–331. leukemia HL-60 cells. Chin. J. Immunol. 16, 8–11. Wang, Q., Ji, Y., Wang, X., Evers, B.M., 2000. Isolation and molecular characterization of the 5V-upstream region of the human TRAIL gene. Biochem. Biophys. Res. Commun. 276, 466–471.