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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 transcription‐starting exon. This initiation site marks an alternative starting exon, designated 1a. Exons 1 and 1a were used simultaneously as transcription‐starting 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 Ca 2+‐ and Mg 2+/Mn 2+‐dependent endonucleolytic cleavage to produce oligonucleotides with 5′‐phosphoryl‐ and 3′‐hydroxy termini ,. 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 -. However, the presence of the enzyme in mammalian tissues other than the digestive organs - 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.
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 cytoplasm and nucleus of such cells.
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 ,. Recently, we demonstrated that an abrupt elevation of serum DNase I activity occurs within ∼ 3 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.
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 3 h after completion of the PCI procedure, returning to basal levels by 12–24 h, in a manner similar to in AMI.
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 2 includes the translation initiation codon (ATG) and the first exon is believed to include only the 5′‐UTR of the mRNA ,. However, comparison of the sequence of the 5′‐UTR of human pancreatic DNASE1 mRNA reported previously with the human genomic sequence showed that the 5′‐terminal 15 nucleotides of the 5′‐UTR are not found in the human genomic sequence , whereas the 3′ segment of 143 nucleotides in the 5′‐UTR matches the genomic sequence.
Thus, the transcription initiation site of exon 1 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 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.
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 ligase‐mediated and oligo‐capping RACE 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 1 reported previously matched with the sequence deposited in GenBank (Accession no.
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 1 comprises 243 bp. 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. +1 above the sequence indicates the 5′‐end of exon 1.
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. Exon 1 (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 1 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 1. −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. Human DNase I activity is mainly demonstrable in pancreas, alimentary tract and pituitary , , 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. 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 1 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 to the beginning of exon 1, as shown in. 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 1.
More interestingly, the products lacked the sequence between positions +1 and +155 in exon 1. 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 1 had AG. Therefore, the splice sites seem to be compatible with a splicing junction. 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 confirm the splicing junction between exon 1 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 8. DNA fragments of different sizes were amplified from the RNA of QGP‐1 cells and pancreas. Determination of the nucleotide sequences of the RT‐PCR products revealed that the sequence between positions +156 and +243 in exon 1 was linked with that of exon 1a ∼ 12 kb upstream of exon 1 in both QGP‐1 cells and pancreas. In addition, another RT‐PCR with a primer corresponding to the 5′‐terminus of exon 1 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 1 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. 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 3.
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 previously , 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.
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 8 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 1 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 1. The thick straight lines represent the intron sequence.
+1 indicates the position of the transcription start site of exon 1. Dashed v‐shaped lines in RT‐PCR amplified fragments A–J indicate regions that are removed by splicing, whereas a dashed line in exon 7 of fragment C represents a deletion of 39 bp. The thick lines indicate intron 6 of 75 bp in fragments B and C, 3′ portion of 35 bp in intron 5 in fragments C and D, and 3′ portion of 25 bp in intron 6 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. Quantitative real‐time RT‐PCR was performed using each distinct starting exon‐specific primer and a common reverse primer complementary to exon 2 to determine the relative abundance by comparing the copy number of transcripts containing exon 1a with the number starting from exon 1.
The abundance of transcripts starting from exon 1 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 1 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.
The findings indicate that transcripts starting from either exon 1a or exon 1 are translated to produce intact DNase I protein. 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 1 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 1. 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 1. 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 1 between positions +156 and +243. The resulting DNase I activity in the medium secreted from each transfected cells was normalized by coexpressed β‐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. Characterization of the promoter region of exons 1a and 1 in the human DNase I gene Because 5′‐RACE analysis identified two transcription‐starting exons used in DNASE1, we characterized the promoters that regulate transcription of the DNASE1 messages containing exons 1 or 1a. To examine promoter activity in the 5′‐flanking region of exon 1 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. 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 1 in DNASE1.
Deletion of the upstream end of the 5′‐flanking region of exon 1 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 1. Summary of relative luciferase activities of the reporter constructs containing the different length of 5′ upstream sequence of the human DNASE1 exon 1 (A) or exon 1a (B). The different length of 5′ upstream sequence of the human DNASE1 exon 1 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 1. 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→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 µg 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. 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 1 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 , 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 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. 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. 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.
The oligonucleotide 90,51 probe produced a major up‐shifted band when the probe was incubated with the nuclear extracts (lanes 1 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 2 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 3 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 Sp1‐binding 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.