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

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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 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. 转载 doi:10.1016/j.gene.2003.12.025 中国科技论文在线 http://www.paper.edu.cn 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).
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