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

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. 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. rapid amplification of cDNA ends nucleotide glyceraldehyde-3-phosphate dehydrogenase hepatic leukemia factor nerve growth factor analysis of variance reverse transcription enhanced green fluorescent protein Translocation Ets Leukemia CCAAT/enhancer-binding protein β. Apoptosis is a genetically controlled cellular response to specific stimuli. It often requires the activation of specific genes (1Johnson Jr., E.M. Deckwerth T.L. Annu. Rev. Neurosci. 1993; 16: 31-46Crossref PubMed Scopus (312) Google Scholar, 2Owens G.P. Cohen J.J. Cancer Metastasis Rev. 1992; 11: 149-156Crossref PubMed Scopus (36) Google Scholar, 3Schwartz L.M. Kosz L. Kay B.K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6594-6598Crossref PubMed Scopus (139) Google Scholar) and can be prevented by inhibitors of RNA and protein synthesis (4Linnik M.D. Zobrist R.H. 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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(9Gottron F.J. Ying H.S. Choi D.W. Mol. Cell. Neurosci. 1997; 9: 159-169Crossref PubMed Scopus (129) Google Scholar, 10Fink K. Zhu J. Namura S. Shimizu-Sasamata M. Endres M. Ma J. Dalkara T. Yuan J. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1998; 18: 1071-1076Crossref PubMed Scopus (198) Google Scholar, 11Clark R.S. Kochanek P.M. Watkins S.C. Chen M. Dixon C.E. Seidberg N.A. Melick J. Loeffert J.E. Nathaniel P.D. Jin K.L. Graham S.H. J. Neurochem. 2000; 74: 740-753Crossref PubMed Scopus (336) Google Scholar, 12Yakovlev A.G. Knoblach S.M. Fan L. Fox G.B. Goodnight R. Faden A.I. J. Neurosci. 1997; 17: 7415-7424Crossref PubMed Google Scholar, 13Eldadah B.A. Yakovlev A.G. Faden A.I. J. Neurosci. 1997; 17: 6105-6113Crossref PubMed Google Scholar, 14Gillardon F. Bottiger B. Schmitz B. Zimmermann M. Hossmann K.A. Brain Res. Mol. Brain Res. 1997; 50: 16-22Crossref PubMed Scopus (136) Google Scholar, 15Namura S. Zhu J. Fink K. Endres M. Srinivasan A. Tomaselli K.J. Yuan J. Moskowitz M.A. J. Neurosci. 1998; 18: 3659-3668Crossref PubMed Google Scholar, 16Clark R.S. Kochanek P.M. Chen M. Watkins S.C. Marion D.W. Chen J. Hamilton R.L. Loeffert J.E. Graham S.H. FASEB J. 1999; 13: 813-821Crossref PubMed Scopus (239) Google Scholar, 17Allen J.W. Knoblach S.M. Faden A.I. FASEB J. 1999; 13: 1875-1882Crossref PubMed Scopus (57) Google Scholar). 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 (18Bittigau P. Sifringer M. Pohl D. Stadthaus D. Ishimaru M. Shimizu H. Ikeda M. Lang D. Speer A. Olney J.W. Ikonomidou C. Ann. Neurol. 1999; 45: 724-735Crossref PubMed Scopus (209) Google Scholar, 19Pohl D. Bittigau P. Ishimaru M.J. Stadthaus D. Hubner C. Olney J.W. Turski L. Ikonomidou C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2508-2513Crossref PubMed Scopus (207) Google Scholar, 20Hu B.R. Liu C.L. Ouyang Y. Blomgren K. Siesjo B.K. J. Cereb. Blood Flow Metab. 2000; 20: 1294-1300Crossref PubMed Scopus (293) Google Scholar, 21Yakovlev A.G. Ota K. Wang G. Movsesyan V. Bao W.L. Yoshihara K. Faden A.I. J. Neurosci. 2001; 21: 7439-7446Crossref PubMed Google Scholar). Furthermore, previous studies, including our own, demonstrated significant increases in the transcriptional activity of the caspase-3 gene during neuronal apoptosis after various stimuli (12Yakovlev A.G. Knoblach S.M. Fan L. Fox G.B. Goodnight R. Faden A.I. J. Neurosci. 1997; 17: 7415-7424Crossref PubMed Google Scholar, 13Eldadah B.A. Yakovlev A.G. Faden A.I. J. Neurosci. 1997; 17: 6105-6113Crossref PubMed Google Scholar, 22Ginham R. Harrison D.C. Facci L. Skaper S. Philpott K.L. Neurosci. Lett. 2001; 302: 113-116Crossref PubMed Scopus (26) Google Scholar, 23Moran J. Itoh T. Reddy U.R. Chen M. Alnemri E.S. Pleasure D. J. Neurochem. 1999; 73: 568-577Crossref PubMed Scopus (88) Google Scholar, 24Harrison D.C. Medhurst A.D. Bond B.C. Campbell C.A. Davis R.P. Philpott K.L. Brain Res. Mol. Brain Res. 2000; 75: 143-149Crossref PubMed Scopus (103) Google Scholar, 25Chen J. Nagayama T. Jin K. Stetler R.A. Zhu R.L. Graham S.H. Simon R.P. J. Neurosci. 1998; 18: 4914-4928Crossref PubMed Google Scholar, 26Kermer P. Klocker N. Labes M. Bahr M. J. Neurosci. 1998; 18: 4656-4662Crossref PubMed Google Scholar, 27Endres M. Namura S. Shimizu-Sasamata M. Waeber C. Zhang L. Gomez-Isla T. Hyman B.T. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1998; 18: 238-247Crossref PubMed Scopus (509) Google Scholar, 28Chiang L.W. Grenier J.M. Ettwiller L. Jenkins L.P. Ficenec D. Martin J. Jin F. DiStefano P.S. Wood A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2814-2819Crossref PubMed Scopus (94) Google Scholar, 29Miller T.M. Moulder K.L. Knudson C.M. Creedon D.J. Deshmukh M. Korsmeyer S.J. Johnson Jr., E.M. J. Cell Biol. 1997; 139: 205-217Crossref PubMed Scopus (360) Google Scholar). 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. The P1Rattus 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 withBamHI 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′-RACE1was performed using Marathon-Ready™ 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. The locations of transcription start sites in the rat caspase-3 gene were determined by a modified primer extension technique (30Yakovlev A.G. Krueger K.E. Faden A.I. J. Biol. Chem. 1995; 270: 6421-6424Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) using an oligonucleotide primer complementary to the previously determined sequence (GenBankTMU58656) from nt 59 to 76 within exon 1. Oligonucleotides were labeled by using [γ-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 This the thermostable Tth to reverse the same RNA after of and with The reaction products were analyzed by in a sequencing from the corresponding region of the rat caspase-3 gene with the same primer in primer extension PC12 and were from the and in modified with (Invitrogen). were at 37 °C in a deletion of the caspase-3 promoter were by of PCR from the promoter between the and HindIII sites of the vector of the cloned DNA were in using the chain termination sequencing reaction. PC12 or were and using and vector (Promega) as an After were and were analyzed for activity by using the and a activity the was of were analyzed with an as previously A.G. Faden A.I. Mol. Chem. 1994; PubMed Scopus Google Scholar). In brief, cellular RNA was isolated by P. N. PubMed Scopus Google and 5 was reverse transcribed with (Invitrogen) in μl of reaction The cDNA was by The of cycles and reaction conditions were to be to a between the of and the of PCR a from to of RNA, as in previously A.G. Faden A.I. Mol. Chem. 1994; PubMed Scopus Google Scholar). to amplify rat caspase-3 cDNA were and cDNA was for cycles of for 30 at 94 °C, annealing for 15 at 55 °C, and primer extension for 45 at 72 cDNA was analyzed in After with were and analyzed by the of were in units as the of PCR to a control from the same RNA The cDNA for was as the to amplify rat cDNA were and cDNA was for cycles at PCR conditions for caspase-3 The of to a corresponding cDNA by DNA sequencing (12Yakovlev A.G. Knoblach S.M. Fan L. Fox G.B. Goodnight R. Faden A.I. J. Neurosci. 1997; 17: 7415-7424Crossref PubMed Google Scholar). were on and in 2 and and on for 5 were by at for 5 min at °C and in were in 5 1 2 5 and 5 by at °C for 30 The was by at for 15 purification of was performed as previously A. A. R. S.M. Mol. 2000; 9: PubMed Scopus Google Scholar). were with 3 of and or and and μl of on a at for 30 The was with μl of μl of 1 of 2 and and of for min at The and were for min with at The were using a in and in sample for After of were by and the were to The were with specific to of clones from the P1 rat genomic DNA library in the of positive hybridization and PCR analysis revealed the in the clone of the transcribed region for the rat caspase-3 gene A the 5′-flanking sequence of the gene was subcloned pZErO-2 plasmid vector (Invitrogen) and The determined sequence was to the A of the sequence with the for rat caspase-3 cDNA and the caspase-3 gene revealed that the cloned rat genomic DNA the first a of and an extended 5′-flanking the of identified a of a element at nt 1 and at nt within the cloned DNA the a was at the of this sequence of the 5′-flanking sequence using in the of a promoter region between nt and The promoter lacks an TATA-box and contains and binding sites. on the sequence previously by determined locations of the transcription start for the caspase-3 gene. The was isolated from rat brain our previous of caspase-3 expression in this A.G. Ota K. Wang G. Movsesyan V. Bao W.L. Yoshihara K. Faden A.I. J. Neurosci. 2001; 21: 7439-7446Crossref PubMed Google Scholar). The primer extension reaction to multiple extension products that to a sequence from to nt upstream of that determined by in The most extended transcription start site for caspase-3 from rat brain can be to a nucleotide 87-bp upstream from the first splicing site. within the rat 5′-flanking sequence relative to this transcription start site A DNA of the cloned 5′-flanking caspase-3 region between and and its deletion by PCR were subcloned the modified The were rat PC12 or human which caspase-3 R. L. H. Yuan J. R. J. Neurosci. Res. 1997; 50: PubMed Scopus Google Scholar, D. N. N. T. N. H. T. Y. J. 2000; 16: PubMed Scopus Google Scholar). demonstrated of expression with the expression from the most extended gene was that by the A vector in which the that contains a promoter region was to expression to of the Furthermore, deletion of a region a cluster of Sp1 elements in a loss of of the from its revealed that the regulatory necessary to expression is located within a between nt −1646 and of this in a in expression to the of the deletion of a region between nt and to a moderate increase in promoter the of a regulatory within the and additional positive elements between nt and A is an that to gene expression by transcriptional as which to of S. Zaman K. H. A. Ratan R.R. Ann. Neurol. 2001; PubMed Scopus Google Scholar). Because the promoter for the rat caspase-3 gene lacks a TATA-box and contains Sp1 binding sites and determined the of mithramycin A on the activity of this A was PC12 and activity was after of in the of or mithramycin A. The addition of mithramycin A to to a inhibition of promoter activity with inhibition at and inhibition at mithramycin A analysis of the region between nt −1646 and for promoter revealed the potential of binding sites for Ets-1-like transcription factors similarity hepatic leukemia factor similarity and transcription factor similarity 5 in binding site for or were then To the site a nucleotide within a binding site was using the PCR in the binding site were with in the site were with A and were in PC12 and activity was Results demonstrated that a of or binding sites in a in the caspase-3 promoter activity 5 To a role of the additional were in the of the promoter with nt The were in PC12 cells. Results of the that a deletion of the binding region nt −1646 to was for loss of promoter activity 5 To the binding of the Ets-1-like element with of the of transcription factors examined the binding of the protein to the corresponding region of the gene. corresponding to a or a of the Ets-1-like element in the rat caspase-3 gene were to and with protein isolated from rat brain. After were and analyzed by using specific to the protein Results that a a binding site was to with this of the transcription factor examined the of growth factor on caspase-3 in PC12 cells. the in were with for 5 followed by of and in a significant in caspase-3 after The addition of to prevented an increase in caspase-3 caspase-3 in the of To the activity of the caspase-3 promoter is by growth factor PC12 were with and activity was after with or or Results demonstrated an increase in activity in the of and with activity in the of or A DNA the promoter sequence from nt −1646 to was then upstream from the enhanced fluorescent protein gene in the vector and the was PC12 cells. revealed of green in cells. In was in with apoptotic after followed by of growth conditions To that activation of the caspase-3 promoter to an increase in protein examined protein expression in PC12 as a of after growth factor analysis that protein were after of The human caspase-3 gene from nt to within the sequence of the 5′-flanking human caspase-3 gene sequence nt to using in the of a promoter region between nt and of the rat and human using within a analysis of the human promoter using demonstrated to the rat promoter, lacks an TATA-box contains and binding sites. and sequence that a from nt to within a with the cloned rat caspase-3 gene regulatory This is located upstream from the transcription start site Previous studies demonstrated of caspase-3 gene activity during the course of apoptosis in neuronal the of gene activation (12Yakovlev A.G. Knoblach S.M. Fan L. Fox G.B. Goodnight R. Faden A.I. J. Neurosci. 1997; 17: 7415-7424Crossref PubMed Google Scholar, 13Eldadah B.A. Yakovlev A.G. Faden A.I. J. Neurosci. 1997; 17: 6105-6113Crossref PubMed Google Scholar, 24Harrison D.C. Medhurst A.D. Bond B.C. Campbell C.A. Davis R.P. Philpott K.L. Brain Res. Mol. Brain Res. 2000; 75: 143-149Crossref PubMed Scopus (103) Google Scholar, 25Chen J. Nagayama T. Jin K. Stetler R.A. Zhu R.L. Graham S.H. Simon R.P. J. Neurosci. 1998; 18: 4914-4928Crossref PubMed Google Scholar, 26Kermer P. Klocker N. Labes M. Bahr M. J. Neurosci. 1998; 18: 4656-4662Crossref PubMed Google Scholar, 27Endres M. Namura S. Shimizu-Sasamata M. Waeber C. Zhang L. Gomez-Isla T. Hyman B.T. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1998; 18: 238-247Crossref PubMed Scopus (509) Google Scholar, 28Chiang L.W. Grenier J.M. Ettwiller L. Jenkins L.P. Ficenec D. Martin J. Jin F. DiStefano P.S. Wood A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2814-2819Crossref PubMed Scopus (94) Google Scholar, 29Miller T.M. Moulder K.L. Knudson C.M. Creedon D.J. Deshmukh M. Korsmeyer S.J. Johnson Jr., E.M. J. Cell Biol. 1997; 139: 205-217Crossref PubMed Scopus (360) Google Scholar). In this report identified the rat caspase-3 gene promoter, determined its structure, and mechanisms of its The was to a general that differential regulation of the caspase-3 gene may for the control of neuronal of a P1 rat genomic DNA library in the of a 5′-flanking region of the rat caspase-3 gene. analysis of the nucleotide sequence from this with a promoter located between nt and relative to the most upstream transcription start site. This promoter a TATA-box a cluster of Sp1 binding sites. The of a TATA-box is a of genes L. D. FASEB J. 1992; PubMed Scopus Google Scholar). In elements located a transcription start of transcription. In the of the rat caspase-3 identified a cluster of transcription start sites. The most upstream transcription start site identified is located in the of a Sp1 analysis that a promoter for human caspase-3 with the identified rat promoter, lacks an and contains multiple Sp1 and binding site. that the 5′-flanking sequence of the human gene with the rat gene regulatory suggest that regulatory of the caspase-3 gene between A demonstrated that the inhibition of Sp1 and binding by mithramycin A neuronal apoptosis by or DNA S. Zaman K. H. A. Ratan R.R. Ann. Neurol. 2001; PubMed Scopus Google Scholar). The that mithramycin A and its be for the of with apoptosis. In the examined an of mithramycin A on the activity of the rat caspase-3 gene Results demonstrated that the addition of this to a significant inhibition of caspase-3 promoter activity. of caspase-3 and protein for models of neuronal apoptosis (12Yakovlev A.G. Knoblach S.M. Fan L. Fox G.B. Goodnight R. Faden A.I. J. Neurosci. 1997; 17: 7415-7424Crossref PubMed Google Scholar, 13Eldadah B.A. Yakovlev A.G. Faden A.I. J. Neurosci. 1997; 17: 6105-6113Crossref PubMed Google Scholar, 24Harrison D.C. Medhurst A.D. Bond B.C. Campbell C.A. Davis R.P. Philpott K.L. Brain Res. Mol. Brain Res. 2000; 75: 143-149Crossref PubMed Scopus (103) Google Scholar, 25Chen J. Nagayama T. Jin K. Stetler R.A. Zhu R.L. Graham S.H. Simon R.P. J. Neurosci. 1998; 18: 4914-4928Crossref PubMed Google Scholar, 26Kermer P. Klocker N. Labes M. Bahr M. J. Neurosci. 1998; 18: 4656-4662Crossref PubMed Google Scholar, 27Endres M. Namura S. Shimizu-Sasamata M. Waeber C. Zhang L. Gomez-Isla T. Hyman B.T. Moskowitz M.A. J. Cereb. Blood Flow Metab. 1998; 18: 238-247Crossref PubMed Scopus (509) Google Scholar, 28Chiang L.W. Grenier J.M. Ettwiller L. Jenkins L.P. Ficenec D. Martin J. Jin F. DiStefano P.S. Wood A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2814-2819Crossref PubMed Scopus (94) Google Scholar, 29Miller T.M. Moulder K.L. Knudson C.M. Creedon D.J. Deshmukh M. Korsmeyer S.J. Johnson Jr., E.M. J. Cell Biol. 1997; 139: 205-217Crossref PubMed Scopus (360) Google Scholar). In this using PC12 that growth factor in an increase in caspase-3 and that this is with the activation of the promoter in apoptosis. Results of the deletion analysis demonstrated that the caspase-3 promoter is necessary for the expression of the activity of the isolated promoter is of the gene 5′-flanking region revealed that major elements necessary for significant transcriptional activity located between −1646 and The of elements relative to the promoter and transcription start sites that to an analysis revealed the in this region of binding sites for transcription factors including and demonstrated an important role for an Ets element in the regulation of the caspase-3 promoter activity. binding of the of the Ets to this regulatory element was demonstrated in this identified transcription factors the same DNA binding of a specific for control of the caspase-3 gene activity requires additional The Ets of transcription factors is to control the expression of genes for cellular and 2000; PubMed Scopus Google Scholar). expression of Ets genes is with development of the 2000; PubMed Scopus Google Scholar). 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