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J. Biol. Chem., Vol. 277, Issue 10, 8273-8278, March 8, 2002

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Identification and Functional Analysis of the Rat Caspase-3 Gene Promoter^*

Wenfang Liu, Geping Wang, and Alexander G. Yakovlev

From the Department of Neuroscience, Georgetown University Medical Center, Washington, D. C. 20007

Received for publication, November 9, 2001, and in revised form, December 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Caspase-3 is the major effector in apoptosis triggered by various stimuli. Previous studies demonstrated a significant increase in transcriptional activity of the caspase-3 gene during neuronal apoptosis. Recent findings suggest that differential expression of the caspase-3 gene may underlie the regulation of apoptotic susceptibility during brain development and after acute injury to the mature brain. We identified and cloned the rat caspase-3 gene promoter, determined its structure, and examined its regulation during a course of apoptosis in PC12 cells. Results demonstrate that this promoter lacks a TATA-box and contains a cluster of Sp1 elements and multiple transcription start sites. The first identified transcription start site is located 87-bp upstream from the first splicing site. A role of Sp1 elements in the regulation of caspase-3 promoter activity is demonstrated by the inhibition of Sp1 binding using mithramycin A. Results of deletion analysis show that an Ets-1-like element located between nucleotides 1646 and 1632 relative to the most extended transcription start site is necessary to achieve sustained transcriptional activity. Homology analysis revealed that the 5'-flanking region of the human caspase-3 gene exhibits significant similarity to a regulatory region of the rat gene.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Apoptosis is a genetically controlled cellular response to specific stimuli. It often requires the activation of specific genes (1-3) and can be prevented by inhibitors of RNA and protein synthesis (4-7).

Of the 14 caspases identified in mammals, caspase-3 appears to be the major effector in neuronal apoptosis triggered by various stimuli. The first strong evidence supporting the specific role for this protease in neuronal apoptosis comes from studies on mice deficient in caspase-3 in which brain development is profoundly altered (8). A role for caspase-3 in neuronal loss was subsequently established using semispecific peptide caspase inhibitors in various models of apoptosis triggered by ischemic or traumatic injury in vivo and in vitro (9-17).

Because the activation of caspases, and caspase-3 in particular, appears to be a major factor for the execution of neuronal apoptosis, the evaluation of upstream modulatory mechanisms is important for understanding the regulation of the apoptotic process. Recent findings suggest that differential expression of the caspase-3 gene may underlie the regulation of apoptotic susceptibility during brain development as well as after acute injury to mature brain (18-21). Furthermore, previous studies, including our own, demonstrated significant increases in the transcriptional activity of the caspase-3 gene during neuronal apoptosis after various stimuli (12, 13, 22-29). Therefore, in addition to the regulation of proteolytic activation, a potential rate-limiting step leading to regulation of caspase-3 activity may be the transcription of this gene. This report provides the first information on the rat caspase-3 gene control region and general transcription factors required for transcriptional activity of this gene.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of the Rat Caspase-3 Gene Promoter-- The P1 Rattus norvegicus genomic DNA library (IncyteGenomics, cat. no. P1-5538) was screened by PCR using 5'-AAATTCAAGGGACGGGTCAT-3' and 5'-ATTGACACAATACACGGGATCTGT-3' primers derived from the coding region of rat caspase-3 cDNA (30 cycles at 94 °C, 30 s; 55 °C, 15 s; and 72 °C, 45 s).

DNA from a PCR-positive P1 clone was isolated, digested with BamHI or HindIII restriction endonucleases, and subcloned in pZErO-2 plasmid vector (Invitrogen). One hundred recombinant DNA clones were screened using the ³²P-labeled oligonucleotide 5'-GGGCGGTAGGCTGCTGATGC-3' corresponding to exon 1 of the rat caspase-3 gene. Hybridization was performed at moderate stringency (6× saline/sodium phosphate/EDTA, 0.2% SDS at 37 °C). Individual positive clones were isolated after an additional round of colony purification at the same hybridization stringency. Isolated clones were then analyzed by restriction mapping with numerous restriction endonucleases and PCR followed by sequencing using the chain termination reaction.

5'-RACE-- 5'-RACE¹ was performed using Marathon-Ready^TM cDNA from Sprague-Dawley rat brain (BD Biosciences, CLONTECH) according to the manufacturer's recommendations. In brief, 5 µl of a cDNA sample were subjected to the first round of PCR amplification using the adaptor primer 1 and the antisense caspase-3 primer 5'-CATTTCTTTAGTGATAAAA-3'. After amplification for 30 cycles (94 °C, 30 s; 55 °C, 15 s; and 72 °C, 2 m), the reaction products were diluted 20-fold with water, and 3 µl were then reamplified for 30 cycles using the same reaction conditions with nested primer adaptor primer 2 and the antisense caspase-3 primer 5'-ATCATGGGATCTGTTTCTTT-3'. The longest detected PCR products were then cloned in the pCR2.1 TA-cloning vector (Invitrogen) and sequenced by the chain termination reaction.

Primer Extension Analysis-- The locations of transcription start sites in the rat caspase-3 gene were determined by a modified primer extension technique (30) using an oligonucleotide primer complementary to the previously determined sequence (GenBank^TM U58656) from nt 59 to 76 within exon 1. Oligonucleotides were labeled by using [-³²P]ATP (6,000 Ci/mmol, Amersham Biosciences) and T4 polynucleotide kinase (Promega, Inc.). Twenty micrograms of yeast (control) or rat brain RNA, pretreated with RNase-free DNase I (Promega), were reverse transcribed with 15 pmol of phosphorylated extension primer and 5 units of thermostable Tth DNA polymerase (Promega) in 30 µl of reaction mixture, as recommended by the manufacturer for reverse transcription. To amplify the signal, 30 cycles of 1 min at 95 °C for denaturation and 5 min at 70 °C for primer annealing and extension were performed. This process permits the thermostable Tth to reverse transcribe the same RNA templates after subsequent denaturing of RNA/DNA hybrids and reannealing with oligonucleotide. The reaction products were analyzed by electrophoresis in denaturing 6% polyacrylamide gels beside a sequencing ladder from the corresponding region of the rat caspase-3 gene obtained with the same primer used in primer extension procedures.

Cell Culture, Transfection, and Luciferase Assay-- PC12 and HeLa cells were obtained from the ATCC and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen). Cultures were maintained at 37 °C in a humidified 5% CO₂-containing atmosphere.

Progressive deletion constructs of the caspase-3 promoter were engineered by unidirectional cloning of PCR fragments from the promoter between the NheI and HindIII sites of the reporter luciferase vector pGL3-basic (Promega). Nucleotide sequences of the cloned DNA fragments were confirmed in each case using the chain termination sequencing reaction.

PC12 or HeLa cells were seeded into 6-well plates and transiently transfected using Mirus TransIT-100 reagent (Panvera). Transfections included reporter plasmids and pRSV-galactosidase reporter vector (Promega) as an internal control. After 24 h, cells were harvested and lysates were analyzed for luciferase activity by using the Enhanced Luciferase Assay kit (Analytical Luminescence Laboratory, San Diego) and a TD-20/20 luminometer. For -galactosidase activity the luminescent -galactosidase genetic reporter system (CLONTECH) was used.

Reverse Transcription-PCR-- Levels of mRNA were analyzed with an RT-PCR approach as previously described (31). In brief, total cellular RNA was isolated by acidic phenol extraction (32), and 5 µg was reverse transcribed with M-MLV RT (Invitrogen) in 20 µl of reaction mixture. The resulting cDNA (3 µl) was amplified by PCR. The numbers of cycles and reaction temperature conditions were estimated to be optimal to provide a linear relationship between the amount of input template and the amount of PCR product generated over a wide concentration range: from 0.5 to 10 µg of total RNA, as described in detail previously (31). Primers to amplify rat caspase-3 cDNA were 5'-GGTATTGAGACAGACAGTGG-3' (sense primer) and 5'-CATGGGATCTGTTTCTTTGC-3' (antisense primer). cDNA was amplified for 28 cycles consisting of denaturing for 30 s at 94 °C, annealing for 15 s at 55 °C, and primer extension for 45 s at 72 °C. Amplified cDNA was analyzed in 2% agarose electrophoretic gels. After being stained with ethidium bromide, UV light gel images were captured and analyzed by the Image 1.59 program. Levels of individual mRNA were expressed in arbitrary units as the proportion of individual PCR product mean optical density to a control product mean optical density obtained from the same RNA sample. The cDNA for GAPDH was used as the internal control. Primers to amplify rat GAPDH cDNA were 5'-TAAAGGGCATCCTGGGCTACACT-3' (sense primer) and 5'-TTACTCCTTGGAGGCCATGTAGG-3' (antisense primer). GAPDH cDNA was amplified for 22 cycles at PCR conditions described for caspase-3 cDNA. The identity of each PCR-generated product to a corresponding cDNA has been confirmed by DNA sequencing (12).

Nuclear Extracts-- Rat brains were dissected on ice and immediately homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 1.5 mM MgCl₂, 200 mM sucrose, 0.1% Triton X-100, 2 mM 2-mercaptoethanol, 10 mM NaF, and 0.5 mM phenylmethylsulfonyl fluoride) and incubated on ice for 5 min. Nuclei were pelleted by centrifugation at 1,600 × g for 5 min at 4 °C and washed twice in lysis buffer without Triton X-100. Nuclear proteins were extracted in high salt buffer (420 mM KCl, 50 mM Tris-HCl, pH 7.5, 5 mM MgCl_2, 10% sucrose, 20% glycerol, 1 mM EDTA, 2 mM dithiothreitol, 20 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 µg/ml leupeptin, 5 µg/ml antipain, and 5 µg/ml aprotinin) by gentle mixing at 4 °C for 30 min. The debris was pelleted by centrifugation at 14,000 × g for 15 min.

Binding Assay for Ets-1 Transcription Factors-- Affinity purification of nuclear proteins was performed as described previously (33). Streptavidin-coated Dynabeads (175 µg) (Dynal, Oslo, Norway) were incubated with 3 µg of annealed 5'-biotin-GTAAGCTTACTTCCTAGATTGTGTA-3' and 5'-biotin-TACACAATCTAGGAAGTAAGCTTAC-3'(wild type) or 5'-biotin-GTAAGCTTACGGGGTAGATTGTGTA-3' and 5'-biotin-TACACAATCTACCCCGTAAGCTTAC-3' (mutant) oligonucleotides and 60 µl of phosphate-buffered saline (pH 7.4) on a shaker at room temperature for 30 min. The nuclear extract (250 µg) was preincubated with 125 µl of 10 mM dithiothreitol, 125 µl of 1 µg/µl poly(dI-dC), 0.25 ml of gel shift buffer (GSB) (10 mM HEPES, 40 mM KCI, 2 mM MgCl₂, and 5% glycerol), and 0.5 ml of distilled water for 20 min at room temperature. The extract and oligonucleotide-coated beads were combined for 20 min with gentle agitation at room temperature. The protein-bound beads were separated using a magnetic separator (Dynal), washed once in GSB, and boiled in SDS-protein sample loading buffer for 10 min. After brief centrifugation, aliquots of eluted samples were fractionated by SDS-polyacrylamide gel electrophoresis, and the separated proteins were transferred to nitrocellulose filters. The filters were probed with antibodies specific to Tel proteins (Santa Cruz Biotechnology).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification and Cloning of the Rat Caspase-3 Gene 5'-Flanking Sequence-- PCR-based screening of 105,300 individual clones from the P1 rat genomic DNA library (Incyte Genomics, Inc.) resulted in the isolation of one positive clone. Combined Southern blot hybridization and further PCR analysis revealed the presence in the clone of the entire transcribed region for the rat caspase-3 gene (data not shown). A 2,056-bp BamHI fragment containing the 5'-flanking sequence of the gene was subcloned into pZErO-2 plasmid vector (Invitrogen) and sequenced. The determined sequence was deposited to the GenBank^TM data bank under accession number AF427079 (Fig. 1).

View larger version (65K): [in this window] [in a new window]   Fig. 1.   Nucleotide sequence of the caspase-3 gene promoter. 5'-flanking region of the rat caspase-3 gene was isolated from the P1 genomic DNA library (IncyteGenomics) and sequenced. A core promoter region was predicted using the PromoterInspector program (Genomatrix). The predicted core promoter region is highlighted in black. Binding sites for transcription factors were predicted using the MatInspector program (Genomatrix). Orientation of predicted binding sites for transcription factors is shown with arrows. Partial sequence of a repeated element B3 is shown in lowercase. Numbering is relative to the first transcription start site identified as described in the legend to Fig. 2. The intronic sequence (underlined) is predicted according to results of an alignment with rat caspase-3 cDNA and the mouse caspase-3 gene.

A 3-way alignment of the obtained sequence with the 5'-end sequences for rat caspase-3 cDNA and the mouse caspase-3 gene revealed that the cloned rat genomic DNA fragment contained the first exon, a part of intron 1, and an extended 5'-flanking region. Using REPEAT software (WebGene, the Institute of Advanced Biomedical Technologies) we identified a portion of a B3 repeated element beginning at nt 1 and ending at nt 88 within the cloned DNA fragment. Using the WWWCPG program (WebGene) a CpG island was localized at the 3'-end of this sequence (nt 1336-2056).

Analysis of the 5'-flanking sequence using PromoterInspector software (Genomatix Software GmbH, Munich) resulted in the prediction of a core promoter region between nt 1637 and 1932. The predicted core promoter lacks an apparent TATA-box and contains six Sp1- and one GC-box binding sites.

Determination of a Transcription Start Site-- Based on the sequence previously obtained by 5'-RACE (GenBank^TM U58656), we next determined locations of the transcription start points for the caspase-3 gene. The target mRNA was isolated from fetal rat brain because our previous results showed high levels of caspase-3 mRNA expression in this tissue (21). The primer extension reaction gave rise to multiple extension products that corresponded to a sequence extending from 6 to 12 nt upstream of that determined by us in 5'-RACE experiments (Fig. 2). The most extended transcription start site for caspase-3 mRNA from rat fetal brain can thus be assigned to a G nucleotide 87-bp upstream from the first splicing site. All positions within the rat 5'-flanking sequence have been indexed relative to this +1 transcription start site (Fig. 1).

View larger version (52K): [in this window] [in a new window]   Fig. 2.   Primer extension analysis for the rat caspase-3 gene. The locations of the transcription start sites in the gene were determined by a modified primer extension technique using RNA from rat brain (R). Yeast RNA was used as a negative control (Y). The reaction products were analyzed by electrophoresis in denaturing 6% polyacrylamide gels beside a sequencing ladder (GATC) from the corresponding region of the gene obtained with the same primer used in primer extension procedures. Locations of transcription start sites mapped by this procedure are highlighted by arrows.

Functional Characterization of the Rat Caspase-3 5'-Flanking Region-- A DNA fragment of the cloned 5'-flanking caspase-3 region between positions 1841 and +88 and its serial deletion derivatives obtained by PCR were subcloned into pGL3-Basic vector, encoding the modified firefly luciferase (Promega). The resulting constructs were transiently transfected into rat PC12 or human HeLa cells, which normally express caspase-3 (34, 35). Both cell lines demonstrated similar profiles of luciferase expression upon transfection with the reporter constructs (Fig. 3). Thus, luciferase expression from the most extended reporter gene (-1841/88) was ~40-fold greater than that produced by the promotorless pGL3-Basic. A vector in which the 237/88-nt fragment that contains a predicted core promoter region was placed to drive luciferase expression failed to express detectable levels of the enzyme. Furthermore, deletion of a region containing a cluster of Sp1 elements (construct 1646/67) also resulted in a loss of luciferase expression.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Deletion analysis of caspase-3 promoter activity in PC12 and HeLa cells. The 5'- and 3'-end points of each deletion construct are indicated. Each construct was transiently transfected into cultured cells and assayed for luciferase activity. Transfections were performed in 3-9 individual experiments. Luciferase activity is normalized to -galactosidase activity and expressed as percent of full-length promoter activity. The results are shown as the mean ± S.D.

Progressive deletions of the 1841/88 construct from its 5'-end revealed that the essential regulatory element(s) necessary to sustain luciferase expression is located within a 43-bp segment between nt 1646 and 1603. Deletion of this segment resulted in a decrease in luciferase expression to the level of the promotorless control. Further deletion of a region between nt 1603 and 1542 led, however, to a moderate increase in promoter activity, suggesting the presence of a negative regulatory element(s) within the 1603/1542 segment and additional positive elements between nt 1542 and 1037 (Fig. 3).

Mithramcyin A Inhibits Caspase-3 Gene Promoter Activity-- Mithramycin A is an aureolic acid antibiotic that has been shown to selectively inhibit gene expression by displacing transcriptional activators such as Sp1, which bind to GC-rich regions of promoters (36). Because the predicted core promoter for the rat caspase-3 gene lacks a TATA-box and contains several Sp1 binding sites and one GC-box, we next determined the effects of mithramycin A on the activity of this promoter. A full-length (-1841/88) reporter contract was transiently transfected into PC12 cells, and luciferase activity was assayed after incubation of cells in the presence of 25 or 100 nM mithramycin A. The addition of mithramycin A to transfected cells led to a concentration-dependent inhibition of promoter activity with ~40% inhibition at 25 nM and 50% inhibition at 100 nM mithramycin A concentration (Fig. 4).

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of mithramycin A on caspase-3 promoter activity. A wild type (-1646/88) reporter construct was transfected in PC12 cells, and luciferase activity was determined after 24 h of incubation in the absence or presence of 25 or 100 nM mithramycin A. Luciferase activity is normalized to -galactosidase activity and expressed as mean percent of control ± S.D. (n = 3). *, p < 0.01, compared with activity in the absence of mithramycin A by ANOVA, followed by Dunnett's test.

A Role of the Ets-1 Binding Site in Regulation of the Caspase-3 Promoter-- MatInspector-based analysis of the region between nt 1646 and 1603 (critical for promoter activity) revealed the potential presence of binding sites for Ets-1-like transcription factors (core similarity 1.000), hepatic leukemia factor (HLF, core similarity 1.000), and CEBPB transcription factor (core similarity 0.985) (Fig. 5A). Reporter plasmids containing mutations in each individual binding site for Ets-1, HLF, or CEBPB were then constructed. To alter the HLF site a T nucleotide within a consensus binding site (ATTRYGTAAY) was deleted using the PCR approach. Four essential bases 5'-GGAA-3' in the Ets-1 binding site were replaced with 5'-CCCC-3'. 5'-GAAA-3' in the CEBPB site were replaced with 5'-CGGG-3'.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Analysis of a caspase-3 gene regulatory region. A, structure of an essential caspase-3 gene regulatory region. Position and orientation of possible binding sites for Ets-1-like, HLF, and CEBPB transcription factors are indicated with arrows. Nucleotides mutated in experiment B are underlined. Positions of 5'-end nucleotides in deletion constructs in experiment C are marked with asterisks. B, mutant reporter constructs were produced for each individual transcription factor's binding site. Mutated nucleotides are underlined in A. A wild type (WT) and three mutant reporter constructs (-1646/88) were transfected in PC12 cells, and luciferase activity was assayed as described under "Experimental Procedures." Results are expressed as mean percent of wild type promoter activity ± S.D. (n = 4). *, p < 0.05, compared with wild type activity by ANOVA, followed by Dunnett's test. C, deletion of the Ets-1 element leads to loss of promoter activity. The 5'- and 3'-end points of each deletion construct are indicated. Each construct was transiently transfected into PC12 cells and assayed for luciferase activity. Results are expressed as mean percent of (-1646/88) promoter activity ± S.D. (n = 3).

A wild type and three mutant reporter constructs (-1646/88) were separately transfected in PC12 cells, and luciferase activity was assayed. Results demonstrated that a mutation of Ets-1 but not HLF or CEBP binding sites resulted in approximately a 4-fold decrease in the caspase-3 promoter activity (Fig. 5B).

To confirm a functional role of the Ets-1 element, three additional 10-bp deletions were introduced in the 5'-end of the promoter beginning with nt 1646. The resulting reporter constructs were transfected in PC12 cells. Results of the luciferase assay evidently showed that a small deletion of the Ets-1 binding region (from nt 1646 to 1636) was sufficient for loss of promoter activity (Fig. 5C).

Tel Member of Ets-1 Family Binds to the Caspase-3 Gene Promoter-- To confirm the binding specificity of the predicted Ets-1-like element with members of the Ets-1 family of transcription factors we examined the binding of the Tel protein to the corresponding region of the gene. Double-stranded oligonucleotides corresponding to a wild type or a mutated form of the Ets-1-like element in the rat caspase-3 gene were attached to magnetic beads and incubated with nuclear protein extracts isolated from rat brain. After washing, bound proteins were eluted and analyzed by Western blot using antibodies specific to the Tel protein (Fig. 6). Results showed that a wild type but not a mutant Ets-1 binding site was able to bind with this member of the Ets-1 transcription factor family.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Binding of Tel transcription factor to the regulatory region of the rat caspase-3 promoter. Wild type (WT) or mutant (M) double-stranded biotinylated oligonucleotides corresponding to the Ets-1-like element were attached to streptavidin-coated Dynabeads and incubated with nuclear proteins from rat brain. Bound proteins were eluted and detected by Western blot using anti-Tel antibodies.

Activation of Caspase-3 Promoter by Growth Factor Deprivation-- Using semiquantitative RT-PCR we examined the effect of growth factor deprivation on caspase-3 mRNA levels in differentiated PC12 cells. Thus, the cells in complete medium were treated with 100 ng/ml NGF (Sigma) for 5 days followed by removal of NGF and serum. As shown in Fig. 7A, such treatment caused a significant elevation in caspase-3 mRNA after 6 h. The addition of NGF to serum-free culture medium prevented an increase in caspase-3 mRNA levels. NGF did not significantly affect caspase-3 mRNA levels in the presence of serum.

View larger version (77K): [in this window] [in a new window]   Fig. 7.   Regulation of caspase-3 promoter activity in PC12 cells by trophic factor deprivation. A, caspase-3 mRNA expression in the differentiated cells at 6 h after serum and NGF withdrawal or in complete medium controls. Levels of mRNA were measured by semiquantitative RT-PCR. Levels of caspase-3 mRNA are expressed as the proportion of individual RT-PCR product mean optical density to GAPDH RT-PCR product optical density of the same RNA sample. mRNA content is expressed as a percentage of control ± S.D. (n = 4). *, p < 0.05; #, p < 0.05, compared with control or serum- and NGF-deprived conditions, correspondingly, by ANOVA, followed by Dunnett's test. B, effect of growth factor deprivation on caspase-3 promoter activity. A wild type (-1646/88) reporter construct was transfected in PC12 cells, and luciferase activity was determined after 24 h of incubation with or without serum or NGF. Luciferase activity is normalized to -galactosidase activity and expressed as mean percent of control ± S.D. (n = 6). *, p < 0.05; #, p < 0.01, compared with control or serum- and NGF-deprived conditions, correspondingly, by ANOVA, followed by Dunnett's test. C, a promoter sequence (-1646/88) was inserted upstream from the EGFP reporter in the pEGFP-1 and transfected into PC12 cells. Stable clones were acquired after selection in the presence of 1 mg/ml G418 for 2 weeks and pooled together. Transfected cells were differentiated during 5 days of incubation in the presence of 100 ng/ml NGF followed by the removal of NGF and serum. Intensive green fluorescence was observed in many cells with apoptotic morphology (white arrows) but not in normal differentiated cells (black arrows) under growth factor-deprived conditions. D, PC12 cells, stably transfected with the reporter construct described above, underwent apoptosis after growth factor deprivation. 30-µg aliquots of cytosolic extracts were isolated from control, and growth factor-deprived cells were subjected to 12% SDS-PAGE and transferred to a nitrocellulose filters. The filters were probed with a monoclonal anti-GFP antibody (CLONTECH).

To examine whether the activity of the caspase-3 promoter is affected by growth factor deprivation, PC12 cells were transiently transfected with (-1646/88)-luciferase reporter plasmid, and luciferase activity was assayed after 24 h incubation with or without serum or NGF. Results demonstrated an ~70% increase in luciferase activity in the absence of serum and NGF compared with activity in the presence of serum, NGF, or both (Fig. 7B).

A DNA fragment comprising the promoter sequence from nt 1646 to +88 was then inserted upstream from the enhanced fluorescent protein (EGFP) reporter gene in the promoterless vector pEGFP-1 (CLONTECH), and the obtained construct was stably transfected into PC12 cells. Fluorescent microscopy examination revealed low levels of green fluorescence in transfected cells. In contrast, intensive fluorescence was observed in shrunken cells with apoptotic morphology after NGF-induced differentiation followed by 18 h of incubation under growth factor-deprived conditions (Fig. 7C).

To confirm that activation of the caspase-3 promoter leads to an increase in protein levels, we examined EGFP protein expression in transfected PC12 cells as a function of time after growth factor deprivation. Using Western blot analysis we found that EGFP protein levels were elevated after 6 h of treatment (Fig. 7D).

Rat Caspase-3 Promoter Is Homologous to the Upstream Sequence of the Human Caspase-3 Gene-- The human caspase-3 gene comprises ~21.8 kb from nt 425446 to 447199 within the NT 006256.6 chromosome 4 working draft sequence segment. Analysis of the 5'-flanking human caspase-3 gene sequence (from nt 422092 to 425520; NT 006256.6) using PromoterInspector software (Genomatix) resulted in the prediction of a core promoter region between nt 425068 and 425487. Alignment of the rat and human predicted core promoters using LALIGN showed 61.8% identity within a 225-nt overlap (Fig. 8). Further analysis of the predicted human core promoter using MatInspector demonstrated that, similar to the rat core promoter, it lacks an apparent TATA-box but contains five Sp1- and one GC-box binding sites.

View larger version (44K): [in this window] [in a new window]   Fig. 8.   Alignment of predicted core promoters for rat and human caspase-3 genes. A promoter region within the human caspase-3 gene (GenBankTM NT006256.6) was predicted using PromoterInspector program (Genomatix). Binding sites for transcription factors were predicted using MatInspector program (Genomatix). The positions of the common binding sites in rat and human promoters are indicated in boxes.

Using ClustalW and LALIGN sequence alignment programs we also found that a fragment from nt 422177 to 422942 had 61.7% identity within a 788-nt overlap with the cloned rat caspase-3 gene regulatory region. This fragment is located ~2.5-kb upstream from the transcription start site (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous studies have demonstrated up-regulation of caspase-3 gene activity during the course of apoptosis in neuronal cells; however, the significance of such gene activation remains obscure (12, 13, 24-29). In this report we identified the rat caspase-3 gene promoter, determined its structure, and began examining mechanisms of its regulation. The study was undertaken to test a general hypothesis that differential regulation of the caspase-3 gene may have functional consequences for the control of neuronal death.

Screening of a P1 rat genomic DNA library resulted in the isolation of a 5'-flanking region of the rat caspase-3 gene. Computer analysis of the nucleotide sequence from this fragment predicted, with high probability, a core promoter located between nt 205 and +91 relative to the most upstream transcription start site. This predicted core promoter does not contain a noticeable TATA-box but has a cluster of Sp1 binding sites. The absence of a TATA-box is a notable feature of many housekeeping genes (37). In TATA-less promoters, so-called initiator elements located over a transcription start site, determine basal levels of transcription. In the case of the rat caspase-3 gene, we identified a cluster of transcription start sites. The most upstream transcription start site identified is located in the middle of a Sp1 tandem.

Using homology analysis tools we found that a predicted promoter for human caspase-3 shows 61.8% identity with the identified rat promoter, also lacks an apparent TATA-box, and contains multiple Sp1 and one GC binding site. Moreover, we found that the 5'-flanking sequence of the human gene demonstrates 61.7% identity with the rat gene regulatory region. Overall, these data suggest that regulatory regions of the caspase-3 gene are conserved between these mammalian species.

A recent study has demonstrated that the inhibition of Sp1 and Sp3 binding by mithramycin A inhibited neuronal apoptosis induced by oxidative stress or DNA damage (36). The authors suggested that mithramycin A and its structural analogs might be useful for the treatment of neurological diseases associated with apoptosis. In the present study, we examined an effect of mithramycin A on the activity of the rat caspase-3 gene promoter. Results demonstrated that the addition of this drug leads to a significant concentration-dependent inhibition of caspase-3 promoter activity.

Elevation of caspase-3 mRNA and protein levels has been reported for several models of neuronal apoptosis (12, 13, 24-29). In this study using PC12 cells, we showed that growth factor deprivation also results in an increase in caspase-3 mRNA content and that this event is associated with the activation of the gene-specific promoter in cells undergoing apoptosis.

Results of the deletion analysis demonstrated that the computer-predicted caspase-3 core promoter is necessary for the expression of the luciferase reporter gene; however, activity of the isolated core promoter is extremely low. Functional characterization of the gene 5'-flanking region revealed that major elements necessary for significant basal transcriptional activity are located between positions 1646 and 1603. The location of these elements relative to the predicted core promoter and transcription start sites suggests that they belong to an enhancer region. Computer-based analysis revealed the presence in this region of binding sites for several transcription factors including Ets-1, HLF, and CEBPB. Mutagenesis experiments demonstrated an important role for an Ets element in the regulation of the caspase-3 promoter activity. Direct binding of the Tel member of the Ets family to this regulatory element was also demonstrated in this study. However, because more than 40 identified Ets-like transcription factors share the same consensus DNA binding site, identification of a specific factor(s) responsible for control of the caspase-3 gene activity requires additional experiments.

The Ets family of transcription factors is known to control the expression of genes critical for cellular proliferation, differentiation, and transformation (38). Differential expression of Ets genes is particularly associated with embryonic development of the central nervous system (39). Furthermore, previous studies have implicated Ets transcription factors in apoptosis; however, exact mechanisms remain unclear. Thus, overexpression of Ets-1 and Pu.1 induces apoptosis in various cell types (40-43). Recent findings also implicated Ets-2 in the regulation of oxidant-induced apoptosis (44). The Tel gene has been demonstrated to play an important role in developmental apoptosis of mesenchymal and neural cells (45). Future studies will examine whether a role for Ets family members in neuronal development and apoptosis is based on their ability to regulate caspase-3 gene expression.

	ACKNOWLEDGEMENTS

We thank Drs. K. E. Krueger and V. Soldatenkov for helpful suggestions and discussion of experimental results.

	FOOTNOTES

^* This work was supported by Grant NS38941 (to A. G. Y.) from the NINDS, National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF427079.

To whom correspondence should be addressed: Dept. of Neuroscience, Georgetown University, Research Bldg., WP14, 3970 Reservoir Rd. NW, Washington, D. C. 20007. Tel.: 202-687-1735; Fax: 202-687-0617; E-mail: yakovlev@giccs.georgetown.edu.

Published, JBC Papers in Press, December 28, 2001, DOI 10.1074/jbc.M110768200

	ABBREVIATIONS

The abbreviations used are: RACE, rapid amplification of cDNA ends; nt, nucleotide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HLF, hepatic leukemia factor; NGF, nerve growth factor; ANOVA, analysis of variance; RT, reverse transcription; EGFP, enhanced green fluorescent protein; TEL, Translocation Ets Leukemia; CEBP, CCAAT/enhancer-binding protein ..

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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