Tài liệu Báo cáo khoa học: Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation ppt

Characterization of human deoxyribonuclease I gene (DNASE1) promoters reveals the utilization of two transcription-starting exons and the involvement of Sp1 in its transcriptional regulation Yoshihiko Kominato1, Misuzu Ueki2, Reiko Iida3, Yasuyuki Kawai4, Tamiko Nakajima1, Chikako Makita1, Masako Itoi1, Yutaka Tajima1, Koichiro Kishi1 and Toshihiro Yasuda2 Department of Legal Medicine and Medical Genetics, Gunma University, Japan Division of Medical Genetics and Biochemistry, University of Fukui, Japan Division of Legal Medicine, University of Fukui, Japan Third Division of Internal Medicine, University of Fukui, Japan Keywords alternative splicing; deoxyribonuclease I; genes; promoter; Sp1 Correspondence T Yasuda, Division of Medical Genetics and Biochemistry, Faculty of Medical Sciences, University of Fukui, Eiheiji, Fukui 910-1193, Japan Fax: +81 776 61 8149 Tel: +81 776 61 8287 E-mail: tyasuda@fmsrsa.fukui-med.ac.jp Database The nucleotide sequences reported here have been submitted to the GenBank/ EMBL/DDBJ Data Bank with accession numbers AB188151 and AB188152 (Received 22 March 2006, revised May 2006, accepted 15 May 2006) doi:10.1111/j.1742-4658.2006.05320.x Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown to be altered by physiological and ⁄ or pathological processes However, no information is available on the regulation of DNase I gene (DNASE1) expression in vivo or in vitro We first mapped the transcription start sites of DNASE1 in human pancreas and in the DNase I-producing human pancreatic cancer cell line QGP-1, and revealed a novel site  12 kb upstream of exon 1, which was previously believed to be the single transcriptionstarting exon This initiation site marks an alternative starting exon, designated 1a Exons and 1a were used simultaneously as transcriptionstarting exons in pancreas and QGP-1 cells Promoter assay, EMSA and chromatin immunoprecipitation analysis with QGP-1 cells showed the promoter region of exon 1a in which the Sp1 transcription factor is specifically involved in promoter activity This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes Furthermore, RT-PCR analysis indicated alternative splicing of human DNASE1 pre-mRNA in pancreas and QGP-1 cells Only two transcripts among eight alternative splicing products identified can be translated to produce intact DNase I protein These results suggest that human DNASE1 expression is regulated through the use of alternative promoter and alternative splicing Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an enzyme that preferentially attacks double-stranded DNA by Ca2+- and Mg2+ ⁄ Mn2+-dependent endonucleolytic cleavage to produce oligonucleotides with 5¢-phosphoryl- and 3¢-hydroxy termini [1,2] DNase I is considered to play a major role in the digestion of dietary DNA, because in vertebrates it is secreted by exocrine ⁄ endocrine glands such as the pancreas and parotid gland into the alimentary tract [3–5] However, the presence of the enzyme in mammalian tissues other than the digestive organs [6–8] suggested that it might have other function(s) in vivo; endogenous DNase I has been regarded as a candidate endonuclease responsible for internucleosomal DNA degradation during apoptosis [9] Furthermore, Napirei et al have shown that extracellular (serum) DNase I participates in the chromatin breakdown of necrotic cells, achieved by its diffusion from the extracellular fluid into the Abbreviations AMI, acute myocardial infarction; ChIP, chromatin immunoprecipitation; DNase I, deoxyribonuclease I; DNASE1, DNase I gene; EMSA, electrophoretic mobility shift assay; PCI, percutaneous coronary intervention; SRED, single radial enzyme diffusion 3094 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al cytoplasm and nucleus of such cells [10] In a similar context, DNase I has been postulated to be responsible for the removal of DNA from nuclear antigens at sites of high cell turnover and necrosis, and thus for the prevention of systemic lupus erythematosus [11,12] Recently, we demonstrated that an abrupt elevation of serum DNase I activity occurs within  h of the onset of symptoms in patients with acute myocardial infarction (AMI) and that DNase I activity in serum then exhibits a marked time-dependent decline within 12 h, returning to basal levels within 24 h [13] Moreover, percutaneous coronary intervention (PCI), which is performed to treat patients with stable angina pectoris, offers an in vivo model of mild myocardial ischemia in humans Irrespective of a lack of alteration in levels of other conventional cardiac markers such as creatine kinase isoenzyme MB and cardiac troponin T, serum DNase I levels rose significantly from basal levels by h after completion of the PCI procedure, returning to basal levels by 12–24 h, in a manner similar to in AMI [14] These findings permitted us to suggest that myocardial ischemia rather than injury induces such elevation in serum DNase I activity However, the mechanisms for the elevation of serum DNase I activity induced by ischemia during AMI or PCI remain to be elucidated Delineation of the molecular basis for our observations is essential to evaluate the elevated DNase I activity in the sera of patients with AMI and to validate the use of serum DNase I activity as a new diagnostic marker for the early detection of AMI To elucidate the molecular basis of this phenomenon, it is important to understand the regulatory mechanism of the human DNase I gene (DNASE1) expression Previous molecular–genetic studies have shown that the human DNase I gene consists of at least nine exons spanning > 3.2 kb of genomic DNA at chromosome 16p13.3; exon includes the translation initiation codon (ATG) and the first exon is believed to include only the 5¢-UTR of the mRNA [15,16] However, comparison of the sequence of the 5¢-UTR of human pancreatic DNASE1 mRNA reported previously [15] with the human genomic sequence showed that the 5¢-terminal 15 nucleotides of the 5¢-UTR are not found in the human genomic sequence [16], whereas the 3¢ segment of 143 nucleotides in the 5¢-UTR matches the genomic sequence Thus, the transcription initiation site of exon has not yet been definitively identified To our knowledge, no information is available on the regulation of vertebrate DNASE1 expression in vivo or in vitro, including characterization of the promoter region of the gene and the associated transcriptional factors Therefore, delineation of the transcriptional Promoters of human deoxyribonuclease I gene regulation of human DNASE1 may provide clues to the mechanisms underlying ischemia-induced elevation of DNase I activity in vivo Here, we describe the identification of a novel transcription starting exon, designated as exon 1a, in human DNASE1 and characterization of the promoter regions of the gene; Sp1 transcription factor plays an important role in promoter activity in the 5¢-upstream region of DNASE1 exon 1a Results Mapping of transcription initiation sites in the human DNASE1 gene To identify the transcription initiation sites of DNASE1 in pancreas, 5¢-RACE based on RNA ligasemediated and oligo-capping RACE [17] was performed with cDNA synthesized from pancreas Agarose gel electrophoresis of the 5¢-RACE products showed a slow-migrating major band and several faster-migrating faint bands Alternative splicing occurs in many genes, and so DNA fragments were purified from the major band and cloned into a sequencing vector DNA sequences were determined for seven transformant clones Four clones contained a 476-bp 5¢-RACE DNA product, of which the 3¢ 380 bp were identical to exons 1–3 from the 5¢-end of the reverse primer DN+231 to the 5¢-end of the region where the sequence of exon reported previously [15] matched with the sequence deposited in GenBank (Accession no AC006111) (Fig 1A) The sequence of the 5¢ portion beyond that point was identical to a 96-bp region of the genomic DNA, indicating that the entire sequence of exon comprises 243 bp Human DNase I activity is mainly demonstrable in pancreas, alimentary tract and pituitary [3,18], and most serum DNase I appears to be produced from those tissues Moreover, our survey of DNase I-producing cell lines showed a high level of DNase I activity and its transcripts in QGP-1 cells, which were established by Kaku et al [19] from a human pancreatic islet cell carcinoma (possibly D cells) as a carcinoembryonic antigen-secreting cell line A similar 5¢-RACE was performed with cDNA synthesized from total RNA of QGP-1 cells The DNA sequences of the 5¢-RACE products were determined for five transformant clones Four clones contained a 417-bp 5¢-RACE DNA product that appeared to be a hybrid between exon and the upstream genomic DNA of DNASE1: the sequence of the 3¢ portion in those products was identical to that of exons 1–3 from the 5¢-end of the reverse primer DN+231 to a position +156 relative FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3095 Promoters of human deoxyribonuclease I gene Y Kominato et al A B 3096 Fig The nucleotide sequence of the 5¢-flanking region in the human DNase I gene (A) The sequence located between positions )100 and +250 relative to the 5¢-end in DNASE1 exon +1 above the sequence indicates the 5¢-end of exon Open circles indicate locations of 5¢-ends of the DNASE1 transcripts, determined by 5¢-RACE using cDNA obtained from pancreas The star indicates the 5¢-end of the DNASE1 mRNA reported previously [3] Exon (nucleotides +1 to +243) is indicated by uppercase letters within rectangles The vertical line between positions +155 and +156 represents the splicing junction where exon 1a is ligated to the part of exon between positions +156 and +243 (B) The sequence located between positions )200 and +150 relative to the transcription start site in human DNASE1 exon 1a Upstream counting is done from +1 of exon )11871 above the sequence indicates the 5¢-end of exon 1a The numbers in parentheses demonstrate the position of the corresponding nucleotides relative to the transcription start site in exon 1a Open circles indicate locations of 5¢-ends of the DNASE1 transcripts, determined by 5¢-RACE using cDNA obtained from QGP-1 cells Exon 1a (nucleotides )11871 to )11770) is indicated by uppercase letters within rectangles Several putative transcription factor binding sites were found using TRANSFAC software and are indicated by overbars The underlines represent the locations of the two oligonucleotide probes used for further analysis The position and identity of mutations at )11944 to )11941 are indicated in oligonucleotide m91,50 and reporter construct )197HmSp FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al to the beginning of exon 1, as shown in Fig 1A Beyond that point, however, the sequence of the 5¢ portion showed 100% identity with that of genomic DNA  12 kb upstream of the DNASE1 exon (Fig 1B) More interestingly, the products lacked the sequence between positions +1 and +155 in exon This comparison with the upstream genomic sequence of DNASE1 allowed us to demonstrate the presence of an alternative exon, which we named exon 1a The donor splice site between exon 1a and the subsequent intron had GT, whereas the acceptor site between the subsequent intron and the 5¢-end at position +156 in exon had AG Therefore, the splice sites seem to be compatible with a splicing junction Promoters of human deoxyribonuclease I gene firm the splicing junction between exon and the upstream DNA, RT-PCR was carried out using a primer specific for exon 1a and a reverse primer complementary to the sequence in exon DNA fragments of different sizes were amplified from the RNA of QGP-1 cells and pancreas (Fig 2A) Determination of the nucleotide sequences of the RT-PCR products revealed A Confirmation of utilization of exon 1a as a transcription initiation exon and occurrence of alternative splicing To examine whether exon 1a is used as a transcription starting exon in QGP-1 cells and pancreas, and to con- B Exons 1a DN-110 DN-105 +1 DN-144 DN+385 DN+721 DN-9 DN+89 DN+232 DN+254 DN+785 A 881 bp C FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 895 bp B RT-PCR amplified fragments Fig RT-PCR analysis to detect the transcription-starting exon in DNASE1 of QGP-1 cells and pancreas (A) RT-PCR analysis Total RNA prepared from QGP-1 cells or pancreas was reverse-transcribed with random primer, and the resulting single-strand cDNA was used as a template for PCR analysis The DNASE1 amplification was performed using either distinct starting exon-specific primer DN)110 (left) or DN)144 (right) and a common reverse primer DN+785 complementary to exon of the DNASE1 PCR products were electrophoresed through a 1.5% agarose gel and stained with ethidium bromide The amplified fragments were named A–J A kb Plus DNA Ladder was used as a molecular size marker (B) Splicing patterns of the amplified fragments A–J Nucleotide sequences of these fragments were determined and then compared Schematically represented DNASE1 was aligned with the RT-PCR products amplified, using a set of each starting exon-specific primer (DN)110 or DN)144) and the DN+785 primer, which are represented by arrows Open boxes represent the DNASE1 exons, and a vertical broken line indicates the splicing junction between exon 1a and a portion of exon The thick straight lines represent the intron sequence +1 indicates the position of the transcription start site of exon Dashed v-shaped lines in RT-PCR amplified fragments A–J indicate regions that are removed by splicing, whereas a dashed line in exon of fragment C represents a deletion of 39 bp The thick lines indicate intron of 75 bp in fragments B and C, 3¢ portion of 35 bp in intron in fragments C and D, and 3¢ portion of 25 bp in intron in fragments F and I The number at the right of each RT-PCR product represents the length of the product The scheme also shows the location of the reverse primer DN+89 used in another starting exon-specific PCR and quantitative real-time RT-PCR, the locations of the primers DN)105 and DN)9 used in ChIP assays, the locations of the primers DN+232 and DN+254 utilized in 5¢-RACE, and the locations of the primers DN+385 and DN+721 used in 3¢-RACE 877 bp D 841 bp E 806 bp F 755 bp G 730 bp H 929 bp I 789 bp J 764 bp 3097 Promoters of human deoxyribonuclease I gene Y Kominato et al that the sequence between positions +156 and +243 in exon was linked with that of exon 1a  12 kb upstream of exon in both QGP-1 cells and pancreas (Fig 2B) In addition, another RT-PCR with a primer corresponding to the 5¢-terminus of exon and the same reverse primer, followed by sequence determination, showed that DNA fragments derived from exon 1, including the DNASE1 full-length and splicing variants, were amplified in QGP-1 cells and pancreas Therefore, these results allow us to conclude that both exon 1a and exon are used simultaneously as transcription-starting exons in QGP-1 cells and pancreas The RT-PCR products of different sizes observed in QGP-1 cells and pancreas seem to arise from alternative splicing The complex patterns of alternatively spliced products are represented schematically in Fig 2B Although the splicing patterns of the DNASE1 transcripts were complex, the transcripts could be classified into two types: first, the full-length transcripts A and H and second, transcripts B–G, I and J that all lack exon Only two DNASE1 transcripts, those corresponding to the amplified products A and H, can be translated to produce intact DNase I protein By contrast, 3¢-RACE using total RNA from QGP-1 cells and pancreas as a template gave a single band with a sequence identical to that reported previ- ously [16], in addition to the observation of a specific cleavage ⁄ polyadenylation site located in the 3¢-flanking region of the gene at position 142 downstream of the stop codon Thus, DNASE1 splicing variants appear to share a common 3¢-UTR Quantitative real-time RT-PCR was performed using each distinct starting exon-specific primer and a common reverse primer complementary to exon to determine the relative abundance by comparing the copy number of transcripts containing exon 1a with the number starting from exon The abundance of transcripts starting from exon was 10-fold higher than that of the transcripts starting from exon 1a in pancreas, whereas it was half in QGP-1 cells To examine whether transcription starting from both exon 1a and exon results in the production of DNase I enzyme, we transfected the expression plasmids ex-pDN1a and ex-pDN1, containing the sequences corresponding to the DNASE1 cDNAs starting from either exon 1a or exon 1, respectively, into COS-7 cells and then determined the levels of DNase I activity secreted into the medium of the cells transfected with each plasmid DNase I activity could be seen in cells transfected with either expression vector, although they differed in levels of expressed DNase I activity (Fig 3) The Relative DNase I activities in the supernatant of COS-7 cells ATG −5 TGA +1 0.5 1.0 coding region ex-pDN +1 exon ex-pDN1 (+1) (+156) exon 1a (-11868) +847 coding region (+243) +1 ex-pDN1a +847 +847 coding region (-11770) Fig Demonstration of the DNase I activities expressed by the DNASE1 expression constructs containing distinct 5¢-UTR regions of DNASE1 mRNA in COS-7 cells DNase I expression vectors, ex-pDN1 and ex-pDN1a, containing the entire 5¢-UTR region of each transcripts derived from exons and 1a, respectively, were constructed and transfected into COS-7 cells Constructs are shown in the left-hand panel The numbers over the diagrams indicate the position of the corresponding nucleotides relative to the translation start site, and the numbers in parentheses below the diagram show the position of the corresponding nucleotides relative to the transcription start site in exon In the expression vector ex-pDN, the Kozak sequence just upstream from the coding region of DNASE1 is contained and indicated by a closed box In the expression vector ex-pDN1, the gray box represents the whole sequence of exon In the expression vector ex-pDN1a, the sequence between +4 and +102 relative to the transcription start site of exon 1a, indicated by the open box, is ligated with the part of exon between positions +156 and +243 The resulting DNase I activity in the medium secreted from each transfected cells was normalized by coexpressed b-galactosidase activity, and is shown in the right panel The mean values and standard deviations were calculated from five independent experiments The activity of the expression plasmid ex-pDN was assigned an arbitrary value of 1.0 The DNase I activities of the cells transfected with ex-pDN1 were statistically significantly lower than those of cells transfected with ex-pDN or ex-pDN1a (P < 0.05) 3098 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al findings indicate that transcripts starting from either exon 1a or exon are translated to produce intact DNase I protein Characterization of the promoter region of exons 1a and in the human DNase I gene Because 5¢-RACE analysis identified two transcriptionstarting exons used in DNASE1, we characterized the promoters that regulate transcription of the DNASE1 messages containing exons or 1a To examine promoter activity in the 5¢-flanking region of exon in DNASE1, we first obtained the )1386M construct by introducing the )1386 to +268 sequence of DNASE1 into the promoterless pGL3–basic vector upstream of the luciferase coding sequence The reporter plasmid was transfected into QGP-1 cells, followed by assay of luciferase activities (Fig 4A) pGL3–promoter vector containing the SV40 promoter and pGL3–basic vector without the promoter sequence were used as positive and negative controls, respectively The relative luciferase activity of the )1386M construct was at least eightfold higher than that of pGL3–basic vector and was half that of pGL3–promoter vector These findings demonstrate the promoter activity of the 5¢-flanking region of exon in DNASE1 Deletion of the upstream end of the 5¢-flanking region of exon from position )1386 to )231, )116, or )78 did not result in any significant change However, deletion of the sequence from position )78 to )54 resulted in the loss of 50% of the luciferase activity These results imply that the )78 to )55 region is required for DNASE1 proximal promoter activity in the 5¢-flanking region of exon Similarly, we obtained the )2081H construct by introducing the )13952 to )11781 sequence of DNASE1 exon 1a relative to the transcription start site of exon into the promoterless pGL3–basic vector upstream of the luciferase coding sequence, followed by transient transfection into QGP-1 cells The relative luciferase activity of the )2081H construct was at least 14-fold higher than that of the pGL3–basic vector and was not inferior to that of the pGL3–promoter vector (Fig 4B), indicating promoter activity of the 5¢-flanking region of exon 1a in DNASE1 These results are consistent with the finding that DNASE1 utilizes two transcription starting exons in QGP-1 cells Deletion of the upstream end of the 5¢-flanking region of exon 1a from position )13952 to )11965 did not result in any significant change However, deletion of the sequence from position )11965 to )11944 elicited an approximate twofold increase in luciferase activity, indicating that negative regulatory elements are present Promoters of human deoxyribonuclease I gene in the )11965 to )11944 region Furthermore, deletion of the upstream end from position )11944 to )11931 resulted in a fivefold decrease in luciferase activity, suggesting that elements important for distal promoter function are contained within the deleted region Inspection of the sequence between )73 and )60 upstream of the transcription start site of exon 1a revealed a putative binding site for Sp1 transcription factor and related proteins, as shown in Fig 1B To evaluate whether the Sp1-binding site in the DNASE1 distal promoter is crucial for expression, a mutated binding site was introduced into the )197H construct, resulting in the loss of 80% of luciferase activity (Fig 4B) The data show that the Sp1 site is important for the DNASE1 distal promoter function involved in transcription from exon 1a To demonstrate whether the sequence between )11944 and )11931 bound Sp1 transcription factor, EMSA was carried out using nuclear extracts prepared from QGP-1 cells (Fig 5) The oligonucleotide 90,51 probe produced a major up-shifted band when the probe was incubated with the nuclear extracts (lanes and 7) Formation of the up-shifted complex, indicated by the arrow, was decreased by the addition of competing unlabeled self oligonucleotide or Sp1 oligonucleotide (lanes and 4), but not by addition of oligonucleotide m90,51 containing the same mutation of the Sp1 site in )197HmSp construct as well as oligonucleotide mSp1 with a mutated Sp1-binding site (lanes and 5) Consistently, formation of the DNA– protein complex was significantly reduced when the oligonucleotide m90,51 probe was incubated with the nuclear extract (lane 6) These observations suggest that an Sp1-like protein binds to the putative Sp1binding site between )73 and )64 relative to the transcription start site of exon 1a To investigate whether Sp1 itself binds to the oligonucleotide 90,51 probe, a supershift assay was performed Although the transcription factor pancreatic duodenal homeobox-1 protein (PDX-1) binds to the sequence C(C ⁄ T) and can heterodimerize with PBX [20], an anti-PDX-1 IgG failed to supershift the DNA–protein complex (lane 8) However, an anti-Sp1 IgG supershifted the DNA– protein complex in association with reduction in the amount of the complex (lane 9), suggesting that Sp1 binds to the putative site in the region from )90 to )51 relative to the transcription start site of exon 1a Because the Sp1-binding site might be recognized by other members of the Sp1 transcription factor family, including Sp2, Sp3, and Sp4 [21], a chromatin immunoprecipitation (ChIP) assay was carried out with antibodies against Sp1 and Sp1-related proteins The precipitated DNA was subjected to PCR with specific FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3099 Promoters of human deoxyribonuclease I gene A −1386 −1053 −78 −231 +1 +268 Y Kominato et al Relative luciferase activities in QGP-1 cells 0.5 B −13952 −13866 −13202 −12068 −11871 −11781 −12395 Relative luciferase activities in QGPI cells −1386M luc −2081H luc −1995H luc −231M luc −1331H luc −137M luc −524H luc −116M luc −197H luc −78M luc −197HmS luc −54M luc −94H luc −33M luc −73H luc −28M luc −60H luc −17M luc −45H luc +1M luc −14H luc pGL3-Basic Vector luc pGL3-Basic Vector luc −1053M luc kb −2.0 kb −1.0 −2.0 −1.0 Fig Summary of relative luciferase activities of the reporter constructs containing the different length of 5¢ upstream sequence of the human DNASE1 exon (A) or exon 1a (B) The different length of 5¢ upstream sequence of the human DNASE1 exon or exon 1a (horizontal bars) were inserted into the upstream of the firefly luciferase coding sequence of pGL3–basic vector The numerals over the diagrams are the nucleotide positions relative to the transcription starting site of exon Constructs are shown in the left-hand panel; construct names are given at the left of the bar and the locations of the inserted fragment are shown The circle represents the CCCC fi AGAG substitution at )11944 and )11941 introduced in reporter construct )197HmSp Each construct as depicted on the left was transiently transfected into QGP-1 cells One microgram of firefly luciferase reporter construct and 0.01 lg of SV40 ⁄ Renilla luciferase were used for each analysis The cells were harvested for firefly and Renilla luciferase after culture for 38 h The obtained firefly luciferase activity was normalized, which is shown in the right-hand panel Mean values and standard deviations were calculated from more than three independent experiments The activity of pGL3–promoter vector containing the SV40 promoter was arbitrary, given the value of 1.0 primers for the endogenous DNASE1 distal promoter region Analysis revealed that Sp1 binds strongly to the DNASE1 promoter, whereas neither Sp2 nor Sp4 bind to the promoter (Fig 6) However, Sp3 seemed to bind weakly to the promoter, although EMSA failed to show a supershifted band with anti-Sp3 IgG (data not shown) These results provide direct evidence for Sp1 binding to the DNASE1 distal promoter The data demonstrate that Sp1 is involved in the DNASE1 distal promoter function in transcription from exon 1a Discussion In previous studies, we were the first to determine the genomic structure of the mammalian DNase I gene; the human gene is located on chromosome 16p13.3, is  kb long and contains nine exons interrupted by eight introns [16] Subsequently, the mouse [22], rat [23] and bovine [24] genes have been shown to be similar to the human gene In this study, we investigated 3100 the upstream region of the human gene 5¢-RACE analysis of QGP-1 cells and pancreas revealed the presence of a novel exon, named exon 1a, 12 kb upstream of the original exon in human DNASE1 Furthermore, because the full-length 5¢-UTR of the transcript from exon was determined, the 5¢ boundary of exon could be confirmed and was not in agreement with earlier studies [15] RT-PCR analysis and promoter assay of the 5¢-upstream regions of both exons and 1a clarified that human DNASE1 utilizes two transcriptionstarting exons simultaneously for expression of the gene Accordingly, the gene organization of human DNASE1 should be corrected to 10 exons interrupted by nine introns spanning  15 kb of genomic DNA Although the DNA region corresponding to exon 1a in human DNASE1 could not be found on inspection of the rat and mouse databases, further investigation might identify an alternative transcription-starting exon in DNASE1 of mammals other than humans Multiple promoters and transcription initiation sites are frequently used to create diversity and flexibility in FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al Fig Sp1 specifically binds to the DNASE1 distal promoter at a site between )91 and )51 relative to the transcription start site of exon 1a EMSA was performed with nuclear extract from QGP-1 cells DNA–protein interaction was investigated using radiolabeled probe 90, 51 (lanes 1–5, 7–8) or m 90, 51 (lane 6) in the presence or absence of a 200-fold molar excess of competing unlabeled oligonucleotides or antibodies as indicated The major shifted complex is indicated by the arrow Oligonucleotides Sp1 and mSp1 contained the wild and mutant types of Sp1 site, respectively (lanes and 5) The nuclear extract was preincubated with anti-PDX-1 (lane 8) or anti-Sp1 IgG (lane 9) A supershifted band with antibody to Sp1 is indicated by the arrowhead Fig ChIP assays of the Sp1-binding status at the endogenous DNASE1 distal promoter in QGP-1 cells with anti-Sp1, -Sp2, -Sp3, and -Sp4 IgG The amplified DNASE1 distal promoter sequences in the input and bound fractions are shown The PCR products of 97 bp were electrophoresed through a 2% agarose gel and stained with ethidium bromide the regulation of gene expression [25] The level of transcription initiation can vary between alternative promoters, the turnover or translation efficiency of Promoters of human deoxyribonuclease I gene mRNA isoforms with different leader exons can differ, and alternative promoter usage can lead to the generation of protein isoforms differing in amino acid sequence Human DNASE1 pre-mRNA is transcribed from different transcription start sites, exons and 1a, resulting in generation of two kinds of gene transcript However, only the sequences of the 5¢-UTR are different between them, because these transcripts share all the other exons, exons 2–9, which contain the entire coding region Thus, use of alternative promoters in DNASE1 results in no generation of protein isoforms The 5¢-UTR of eukaryotic mRNA influences the initiation step of protein synthesis and thereby in part determines the translational efficiency of the transcript [26] In fact, as shown in Fig 3, the translational efficiency of the transcript from exon was about half of that from exon 1a We used genetyx software (GENETYX Corp., Tokyo, Japan) to search for possible secondary structure in the nucleotide sequence of the 5¢-UTR in the transcripts starting from exons and 1a, and found that the 5¢-UTR of the transcript from exon has a higher content of stem-loop structure than does that from exon 1a Because stable stem–loop structures are known to cause significant suppression of translation, the distinctive secondary structures of the 5¢-UTRs in these transcripts could lead to differences in translation efficiency between them Recently, rat DNase I pre-mRNA was reported to be alternatively spliced in the kidney, leading to the generation of two types of transcript of 1.3 and 1.5 kb [27]; the former showed an internal deletion of a 132-bp segment present in exon 1, and these transcripts were produced from the same transcription starting exon Similarly, the human DNASE1 pre-mRNA undergoes extensive alternative splicing in pancreas and QGP-1 cells, resulting in the generation of normal transcripts and many alternative splicing variants initiated from both exons 1a and 1, as shown in Fig 2B Alternative splicing transcripts without exon shift the reading frame and generate a stop codon in exon 4, resulting in no production of active DNase I enzyme Furthermore, differences in splicing patterns between QGP-1 cells and pancreas (Fig 2A) suggest that splicing patterns of DNASE1 pre-mRNA could change in a cell- and ⁄ or tissue-specific manner Therefore, alternative splicing in DNASE1 may potentially serve as a regulatory device to modulate levels of the gene products No information on the regulation of gene expression in vertebrate DNase I gene, such as characterization of the promoter region and transcription factors responsible for gene expression, has been obtained so far In this study, characterization of the promoter region in FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3101 Promoters of human deoxyribonuclease I gene Y Kominato et al the human DNASE1 gene was performed with QGP-1 cells; using promoter deletion analysis we could survey the upstream regions responsible for transcription from exons and 1a (Fig 4), in the latter of which a potential Sp1 site in the promoter contributes to its basal activity EMSA experiments (Fig 5), ChIP assay (Fig 6) and the introduction of mutations in the reporter gene construct revealed that binding of Sp1 to the upstream region of DNASE1 exon 1a controlled the basal distal promoter activity This is the first to be identified as a transcription factor responsible for gene expression of vertebrate DNase I genes Sp1 is the founding member of a growing family of transcription factors that bind and act through GC boxes, being important in the transcription of many cellular genes Sp1 is abundantly distributed in most cell types, irrespective of differences in levels of its expression among different cell types [28] Shiokawa and Tanuma used polyA+ RNA dot blot hybridization to show that DNASE1 is expressed in a wide variety of human tissues [6], being compatible with direct involvement of Sp1 in expression of the gene Furthermore, we demonstrated in preliminary studies that upregulation of distal DNASE1 promoter activity in QGP-1 cells by hypoxia might depend on the Sp1-binding site (Kominato et al., unpublished data) Because levels of DNase I activity are known to differ greatly among human tissues [3,7], it is likely that additional control mechanisms such as alternative splicing and ⁄ or other post-transcriptional regulation are responsible for tissue-specific distribution of DNase I enzyme activity and ligation with the GeneRacer RNA oligo cDNA was synthesized by using Superscript III and the DNASE1specific primer DN+254, the sequence of which was 5¢TAGGTGTCTGGTGCATCCTG-3¢ 5¢-Ends were PCR amplified from these cDNA templates with a primer to the GeneRacer RNA oligo and the DNASE1-specific primer DN+232, the sequence of which was 5¢-TGAGGTTGTC CAGCAGCTTC-3¢ 3¢-RACE was performed using the 3¢-RACE system (Invitrogen Corp.) according to the manufacturer’s instruction Five micrograms of each total RNA was subjected to 3¢-RACE under the same conditions described previously [30] The sequences of the DNASE1specific forward primers used were 5¢-GACACCTTCAA CCGAGAGCC-3¢ (DN+385) and 5¢-ATGCTGCTCCG AGGCGCCGT-3¢ (DN+721) Conditions for these amplifications were 95 °C for min, 40 cycles of 94 °C for min, 55 °C for min, 72 °C for min, followed by incubation at 72 °C for 10 PCR amplifications were performed in a 50 lL reaction mixture containing 10 pmol of each primer, 1.25 units of AmpliTaq Gold (Applied Biosystems, Foster City, CA), 1.5 mm MgCl2, 150 lm dNTP, and 1· buffer After cloning of the PCR-amplified products into a pCR2.1 plasmid vector (Invitrogen Corp.), the nucleotide sequences of the amplified fragments were determined, using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with both M13 forward and reverse primers The sequencing run was performed on a Genetic Analyzer (model 310, Applied Biosystems) and all the DNA sequences were confirmed by reading both strands The nucleotide sequences reported here have been submitted to the GenBank ⁄ EMBL ⁄ DDBJ Data Bank with accession numbers AB188151 and AB188152 Experimental procedures RT-PCR Cells Human pancreatic cancer cell line QGP-1 (JCRB0183) was obtained from Health Science Research Resources Bank (Osaka, Japan) The cells were grown in RPMI-1640 containing 10% fetal bovine serum (Invitrogen Corp., Carlsbad, CA), 50 mL)1 penicillin and 50 lgặmL)1 streptomycin 5Â- and 3Â-RACE analysis 5Â-RACE was performed using the 5¢-GeneRacer kit (Invitrogen Corp.) according to the manufacturer’s instruction Total RNAs isolated from QGP-1 cells using the acid guanidine thiocyanate ⁄ acid phenol method [29] and human pancreas (BD Biosciences Clontech, Palo Alto, CA) were employed for the RACEs Five micrograms of each total RNA was treated with bovine intestinal phosphatase, followed by incubation with tobacco acid pyrophosphatase 3102 cDNA was synthesized from total RNA (2 lg) of QGP-1 cells using random hexamers and Superscript III (Invitrogen Corp.) One and two of 20 lL of the resulting single strand cDNA reaction were used as templates for RT-PCR and quantitative real-time RT-PCR, respectively Similarly, lg of total RNA prepared from human pancreas was also reverse-transcribed with random primer, and the synthesized cDNA was utilized as template in RT-PCR Starting exon-specific amplification of the DNASE1 mRNA was performed using each distinct starting exon-specific primer and the reverse primer DN+785, of which the sequence was 5¢-GCCATAGGCAGCCTGGAAGT-3¢, complementary to exon of DNASE1 The sequences of the starting exon-specific primers were 5¢-GCCTTGAAGTGCTTCTTC AGAGAC-3¢ (DN)144) and 5¢-GCACAACACAGGGAA GCTTGG-3¢ (DN)110), corresponding to the sequence in exons and 1a of the DNASE1, respectively Another starting exon-specific amplification of the DNASE1 message was performed using each distinct starting exon-specific primer FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al and the reverse primer DN+89, of which the sequence was 5¢-ATGTTGAAGGCTGCGATCTTCAG-3¢, complementary to exon of the DNASE1 Conditions for these amplifications were 95 °C for min, 30 cycles of 94 °C for min, 60 °C for min, and 72 °C for min, followed by incubation at 72 °C for 10 Determination of the nucleotide sequences of the amplified fragments were described above Plasmids The different length of 5¢-upstream sequence of exons and 1a in the DNASE1 were PCR-amplified using sequence-specific primers corresponding to the sequence deposited in the GenBank with Accession no AC006111 and subcloned into a firefly luciferase reporter vector, the pGL3–basic vector (Promega, Madison, WI) Nomenclature used for the various reporter constructs is based on the nature of the inserted fragments Letter symbol M reflects the restriction enzyme cleavage site of MlnI at +268 relative to the transcription start site of exon in the DNSAE1, letter symbol H reflects the restriction enzyme cleavage site of HindIII at +90 relative to the transcription start site of exon 1a, whereas numerals indicate the endpoints of the primers used for PCR For example, )197H construct contains the fragment bordered with PCR primer sequence starting at )197 relative to the transcription start site at one end and HindIII site at the other The DNASE1 expression plasmid ex-pDN was constructed by preparing a PCR-amplified fragment containing the entire coding sequence of the human DNASE1 message by using an Expanded High Fidelity PCR system (Roche) with the total RNA from QGP-1 cells and a primer set of the forward primer DN-5R and the reverse primer DN+854X (sequences 5¢-CGGAATTCTCAGGATGAGGGGCATGAAG-3¢ and 5¢-CGCTCGAGGCTGCTCACTTCAGCATCAC-3¢, respectively) and directionally ligating the fragment into the EcoRI ⁄ XhoI site of pcDNA3.1(+) vector (Invitrogen Corp.) The nucleotides underlined in the primer sequences above represent EcoRI or XhoI sites The 5¢-UTRs of the DNASE1 messages were separately amplified by RT-PCR of total QGP-1 RNA with a primer set of the common reverse primer DN+89 and either of the starting exon-specific forward primers DN)243R or DN)183R, the sequences of which were 5¢-CGGAATTCACATTTGCCCCAGGGAAGGTC3¢ and 5¢-CGGAATTCTCCTGCCCAGGACCCGAGG-3¢, respectively The DNASE1 expression plasmids ex-pDN1 and ex-pDN1a, containing the sequences corresponding to the 5¢-UTRs of the transcripts from exons and 1a, respectively, were constructed by use of the overlap extension method [31] with those PCR-amplified fragments and the plasmid ex-pDN The plasmids pD(1) and pD(1a) were constructed by cloning the PCR-amplified fragments obtained with either of the starting exon-specific forward primers DN)144 or DN)110 and the common reverse primer DN+89, respectively, into a pCR2.1 plasmid vector Promoters of human deoxyribonuclease I gene For all the constructs, sequencing was performed over the entire region of the inserted sequences Plasmid DNA was purified by using HiSpeed Plasmid Kit (QIAGEN GmbH, Hilden, Germany) Transfection and luciferase assay Transient transfection experiments into QGP-1 cells were performed with Lipofectamine Plus reagent (Invitrogen Corp.); lg of firefly luciferase reporter and 0.01 lg of pRL-SV40 Renilla luciferase reporter (Promega) were used for each analysis QGP-1 cells were split, 18–24 h prior to transfection, into a six-well tissue culture plate (Becton Dickinson Labware, Franklin Lakes, NJ) at · 105ỈmL)1 At the time of transfection, cells were washed once with Opti-MEM I-reduced serum medium (Invitrogen Corp.) containing neither fetal bovine serum nor l-glutamine Plasmid DNA was suspended in 100 lL of OptiMEM I-reduced serum medium, followed by mixture with lL of Lipofectamine Plus reagent at room temperature for 10 Four microliters of Lipofectamine Plus reagent were diluted in 100 lL of Opti-MEM I-reduced serum medium The two solutions were combined at room temperature for 15 min, followed by the addition of 0.8 mL of Opti-MEM I-reduced serum medium The mixture was then overlaid onto the cells The cells were incubated for h prior to addition of mL of Opti-MEM I-reduced serum medium containing 20% serum to the DNA-containing medium, followed by incubation for 14 h Subsequently, those cells were cultured at 37 °C under either normoxic or hypoxic conditions prior to the measurement of the activities of firefly and Renilla luciferase Cell lysis and luciferase assays were performed using the Dual Luciferase Reporter Assay System (Promega) Light emission was measured by Wallac 1420 ARVOMX (Perkin–Elmer) The values were obtained in relative light units Variations in transfection efficiency were normalized to the activities of Renilla luciferase expressed from cotransfected pRL-SV40 Renilla luciferase reporter vector COS-7 cells were transiently cotransfected by using Lipofectamine Plus reagent (Invitrogen Corp.) with lg of the DNASE1 expression vectors and 0.6 lg of the pSV–b-galactosidase vector (Promega), followed by assay of DNase I and b-galactosidase activities, according to a previously described method [32] Preparation of nuclear extracts and EMSA The nuclear extract and probe were prepared as reported previously [33], and the sequence of oligonucleotide 90,51 probe corresponds to nucleotides )90 to )51 relative to the transcription start site of exon 1a in the DNASE1 EMSA was carried out according to the method described previously [33] The different double-stranded oligonucleotides were obtained by annealing two chemically synthesized strands The double-stranded Sp1 and mSp1 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3103 Promoters of human deoxyribonuclease I gene Y Kominato et al oligonucleotides containing the wild-type and the mutant version of Sp1 site, respectively, were purchased from Santa Cruz Biotechology (Santa Cruz, CA) A 200-fold molar excess of unlabeled competitors over the radiolabeled probe was used for competition analyses For supershift experiments, lL of polyclonal rabbit anti-PDX-1 IgG or anti-Sp1 IgG (Santa Cruz Biotechnology) was added to the nuclear extract, and preincubated on ice for 15 prior to the addition of radiolabeled probe ChIP analysis ChIP assay was performed using chromatin immunoprecipitation assay kit (Upstate, Lake Placid, NY) according to the manufacturer’s instruction Anti-Sp1 IgG was purchased from Upstate, whereas anti-Sp2, anti-Sp3 and anti-Sp4 IgGs were obtained from Santa Cruz Biotechnology PCR was performed to amplify the region between )105 and )9 relative to the transcription start site of exon 1a in DNASE1 using primers DN)105 and DN)9, the sequences of which were 5¢-CCAGCCTGGCTGGTTATCAGTCC-3¢ and 5¢GAGCTCTTCCACACCAGACGCA-3¢, respectively Conditions for these amplifications were 95 °C for min, 37 cycles of 94 °C for min, 65 °C for min, and 72 °C for min, followed by incubation at 72 °C for 10 The PCR products were electrophoresed through a 2% agarose gel and were stained with ethidium bromide The sequences of the amplified fragments were determined as described above Conditions for both amplifications were 95 °C for 10 s, 40 cycles of 95 °C for s, 60 °C for 20 s Quantitative PCR was performed in a 20-lL reaction mixture containing pmol of each primer, 10 lL of 2· SYBR Premix Ex Taq, and of 20 lL single-strand cDNA reactions To determine the absolute copy number of the target transcripts in individual cDNA reaction mixtures, the plasmids pD(1) and pD(1a) were used to generate a calibration curve The plasmid templates were measured using a spectrophotometer, and copy numbers were calculated from the absorbance at 260-nm For each assay, a standard curve was prepared using serial dilutions of template plasmid DNA with known copy numbers in log steps from · 107 copies to · 102 copies in a 2-lL volume All samples to be compared were run in the same assay After completion of the PCR amplification, the data were analysed with the lightcycler ver.3.5 (Roche) The threshold cycle was calculated using the sequence detection software as the cycle number at which the fluorescence of the reporter dye crossed the threshold in log-linear range of PCR The copy numbers of the respective DNASE1 cDNA were quantified by interpolating the results from the threshold cycles Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (15209023, 17659196 to TY, 17659161 to MU and 16209023 to KK) Enzyme assay Enzyme activity of DNase I was determined by the single radial enzyme diffusion (SRED) method as described previously [7] The human specific DNase I activity in the medium secreted from QGP-1 cells was calculated by subtraction of bovine DNase I activity in the fresh medium from the whole DNase I activity in the supernatant of QGP-1 cells DNase I activity was assayed for cell extract prepared from QGP-1 cells by sonication Protein assay was carried out using a protein assay kit (Bio-Rad, Richmond, CA) with BSA as a standard, and 10 lg of the cell extract was applied to the gel plate for SRED method One unit of enzyme activity assayed corresponds to 0.6 ng of purified human DNase I [34] Quantitative real-time RT-PCR Quantitative real-time RT-PCR was performed using LightCyclerTM (Roche Diagnostics GmbH, Mannheim, Germany) and SYBRÒ Premix Ex Taq (TaKaRa, Shiga, Japan) Specific amplifications of individual starting exon-specific variants of the DNASE1 were performed using starting exon-specific forward primer, DN-110 or DN-144, and the common reverse primer DN+89 3104 References Moore S (1981) Pancreatic DNase In The Enzymes, 3rd edn (Boyer PD, ed.), Vol 14, pp 281–296 Academic Press, New York Kishi K, Yasuda T & Takeshita H (2001) DNase I: structure, function, and use in medicine and forensic science Legal Med 3, 69–83 Takeshita H, Mogi K, Yasuda T, Nakajima T, Nakashima Y, Mori S, Hoshino T & Kishi K (2000) Mammalian deoxyribonuclease I are classified into three types: pancreas, parotid and pancreas-parotid (mixed) types, based on differences in tissue concentrations Biochem Biophys Res Commun 269, 481–484 Takeshita H, Yasuda T, Iida R, Nakajima T, Mori S, Mogi K, Kaneko Y & Kishi K (2001) Amphibian DNases I are characterized by a C-terminal end with a unique, cysteine-rich stretch and by the insertion of s serine residue into the Ca2+-binding site Biochem J 357, 473–480 Yasuda T, Iida R, Ueki M, Kominato Y, Nakajima T, Takeshita H, Kobayashi T & Kishi K (2004) Molecular evolution of shark and other vertebrate DNases I Eur J Biochem 271, 4428–4435 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS Y Kominato et al Shiokawa D & Tanuma S (2001) Characterization of human DNase I family endonucleases and activation of DNase c during apoptosis Biochemistry 40, 143–152 Nadano D, Yasuda T & Kishi K (1993) Measurement of deoxyribonuclease I activity in human tissues and body fluids by a single radial enzyme diffusion method Clin Chem 39, 448–452 Napirei M, Ricken A, Eulitz D, Knoop H & Mannherz HG (2004) Expression pattern of the deoxyribonuclease I gene: lessons from the Dnase1 knockout mouse Biochem J 380, 929–937 Mannherz HG, Peitsch MC, Zanotti S, Paddenberg R & Polzar B (1995) A new function for an old enzyme: the role of DNase I in apoptosis Curr Top Microbiol Immunol 98, 161–174 10 Napirei M, Wulf S & Mannherz HG (2004) Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system Arthritis Rheum 50, 1873–1883 11 Napirei M, Karsunky H, Zevnik B, Stephan H, Mannherz HG & Moroy T (2000) Feature of systemic lupus ă ă erythematosus in DNase I-deficient mice Nat Genet 25, 177–181 12 Lachmann PJ (2003) Lupus and desoxyribonuclease Lupus 12, 202–206 13 Kawai Y, Yoshida M, Arakawa K, Kumamoto T, Morikawa N, Masamura K, Tada H, Ito S, Hoshizaki H, Oshima S et al (2004) Diagnostic use of serum deoxyribonuclease I activity as a novel early-phase marker in acute myocardial infarction Circulation 109, 2398–2400 14 Arakawa K, Kawai Y, Kumamoto T, Morikawa N, Yoshida M, Tada H, Kawaguchi R, Taniguchi K, Miyamori I, Kominato Y et al (2005) Serum deoxyribonuclease I activity can be used as a sensitive marker for detection of transient myocardial ischemia induced by percutaneous coronary intervention Eur Heart J 26, 2375–2380 15 Shak S, Capon DJ, Hellmiss R, Marsters SA & Baker CL (1990) Recombinant human DNase I reduces the viscosity of cystic fibrosis sputum Proc Natl Acad Sci USA 87, 9188–9192 16 Yasuda T, Kishi K, Yanagawa Y & Yoshida A (1995) Structure of the human deoxyribonuclease I (DNase I) gene: identification of the nucleotide substitution that generates its classical genetic polymorphism Ann Hum Genet 59, 1–15 17 Maruyama K & Sugano S (1994) Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides Gene 138, 171–174 18 Yasuda T, Takeshita H, Nakajima T, Mogi K, Kaneko Y, Ueki M, Iida R & Kishi K (2002) Abrupt pubertal elevation of DNase I gene expression in human pituitary glands of both sexes FEBS Lett 510, 22–26 19 Kaku M, Nishiyama T, Yagawa K & Abe M (1980) Establishment of a carcinoembryonic antigen-producing Promoters of human deoxyribonuclease I gene 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 cell line from human pancreatic carcinoma Gann 71, 596–601 Dutta S, Gannon M, Peers B, Wright C, Bonner-Weir S & Montminy M (2001) PDX : PBX complexes are required for normal proliferation of pancreatic cells during development Proc Natl Acad Sci USA 98, 1065–1070 Cook T, Gebelein B & Urrutia R (1999) Sp1 and its likes: biochemical and functional predictions for a growing family of zinc finger transcription factors Ann NY Acad Sci 880, 94–102 Peitsch M, Irmler L, French J & Tschopp J (1995) Genomic organization and expression of mouse deoxyribonuclease I Biochem Biophys Res Commun 207, 62–68 Liu QA, Ribecco M, Hou Y, Walker PR & Sikorska M (1997) DNase I primary transcript is alternatively spliced in both normal and apoptic cells: no evidence of up-regulation in apoptosis DNA Cell Biol 16, 911–918 De Marı´ a A & Arruti C (2003) Bovine DNase I: gene organization, mRNA expression, and change in the topological distribution of the protein during apoptosis in lens epithelial cells Biochem Biophys Res Commun 312, 634–641 Ayoubi TAY & van de Ven WJM (1996) Regulation of gene expression by alternative promoters FASEB J 10, 453–460 Kozak M (1992) Regulation of translation in eukaryotic systems Annu Rev Cell Biol 8, 197–225 Basnakian AG, Singh AB & Shah SV (2002) Identification and expression of deoxyribonuclease I (DNase I) alternative transcripts in the rat Gene 289, 87–96 Suske G (1999) The Sp1-family of transcription factors Gene 238, 291–300 Chomczynski P & Sacchi N (1987) Single-step method of RNA isolation by acid guanidinuim thiocyanate–phenol–chloroform extraction Anal Biochem 162, 156–159 Yasuda T, Takeshita H, Iida R, Nakajima T, Hosomi O, Nakashima Y & Kishi K (1998) Molecular cloning of the cDNA encoding human deoxyribonuclease II J Biol Chem 273, 2610–2616 Ho SN, Hunt HD, Horton RM, Pullen JK & Pease LR (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction Gene 77, 51–59 Yasuda T, Takeshita H, Iida R, Kogure S & Kishi K (1999) A new allele, DNASE1*6, of human deoxyribonuclease I polymorphism encodes an Arg to Cys substitution responsible for its instability Biochem Biophys Res Commun 260, 280–283 Hata Y, Kominato Y, Yamamoto F & Takizawa H (2002) Characterization of the human ABO gene promoter in erythroid cell lineage Vox Sang 82, 39–46 Yasuda T, Awazu S, Sato W, Iida R, Tanaka Y & Kishi K (1990) Human genetically polymorphic deoxyribonuclease: purification, characterization and multiplicity of urine deoxyribonuclease I J Biochem (Tokyo) 108, 393–398 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3105 ... Mapping of transcription initiation sites in the human DNASE1 gene To identify the transcription initiation sites of DNASE1 in pancreas, 5¢-RACE based on RNA ligasemediated and oligo-capping... including characterization of the promoter region of the gene and the associated transcriptional factors Therefore, delineation of the transcriptional Promoters of human deoxyribonuclease I gene regulation. .. isoforms The 5¢-UTR of eukaryotic mRNA in? ??uences the initiation step of protein synthesis and thereby in part determines the translational efficiency of the transcript [26] In fact, as shown in