A Bidirectional Promoter Connects the Poly(ADP-ribose) Polymerase 2 (PARP-2) Gene to the Gene for RNase P RNA

STRUCTURE AND EXPRESSION OF THE MOUSE PARP-2 GENE*

Jean-Christophe AméDagger, Valérie Schreiber, Valérie Fraulob§, Pascal Dollé§, Gilbert de Murcia, and Claude P. Niedergang

From the UPR 9003 du CNRS, Laboratoire Conventionné avec le Commissariat à l'Energie Atomique, ULP-Ecole Supérieure de Biotechnologie de Strasbourg, Boulevard Sébastien Brant, F-67400 Illkirch, France and § Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch-Cedex, France

Received for publication, August 28, 2000, and in revised form, December 12, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Poly(ADP-ribose) polymerase 2 (PARP-2) is a DNA damage-dependent enzyme that belongs to a growing family of enzymes seemingly involved in genome protection. To gain insight into the physiological role of PARP-2 and to investigate mechanisms of PARP-2 gene regulation, we cloned and characterized the murine PARP-2 gene. The PARP-2 gene consists of 16 exons and 15 introns spanning about 13 kilobase pairs. Interestingly, the PARP-2 gene lies head to head with the gene encoding the mouse RNase P RNA subunit. The distance between the transcription start sites of the PARP-2 and RNase P RNA genes is 114 base pairs. This suggested that regulation of the expression of both genes may be coordinated through a bi-directional promoter. The PARP-2/RNase P RNA gene organization is conserved in the human. To our knowledge, this is the first report of a RNA polymerase II gene and an RNA polymerase III gene sharing the same promoter region and potentially the same transcriptional control elements. Reporter gene constructs showed that the 113-base pair intergenic region was indeed sufficient for the expression of both genes and revealed the importance of both the TATA and the DSE/Oct-1 expression control elements for the PARP-2 gene transcription. The expression of both genes is clearly independently regulated. PARP-2 is expressed only in certain tissues, and RNase P RNA is expressed in all tissues. This suggests that both genes may be subjected to multiple levels of control and may be regulated by different factors in different cellular contexts.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Poly(ADP-ribosylation) is an immediate post-translational modification of nuclear proteins induced by DNA-damaging agents. At a site of breakage, the enzyme poly(ADP-ribose) polymerase 1 (PARP-1,1 113 kDa) catalyzes the transfer of the ADP-ribose moiety from the respiratory coenzyme NAD+ to a limited number of protein acceptors involved in chromatin architecture (histones H1, H2B, lamin B, and high mobility group [HMG] proteins) or in DNA metabolism (DNA replication factors, topoisomerases including PARP-1) (1-3). Auto- and heteromodification of these proteins are part of an obligatory step of a detection/signaling pathway leading ultimately to the resolution of strand interruptions. A variety of molecular and genetic approaches have been developed this last decade that define unambiguously a critical role of PARP-1 in the cell response to DNA damage and repair and in cell death during the inflammatory process (for review, see Shall and de Murcia (4, 5)).

Although it was assumed for many years that PARP activity was associated with a single protein (now termed PARP-1) displaying unique DNA damage detection properties, the recent discovery of four members of the PARP family has emphasized an unexpected complexity of poly(ADP-ribosylation) reactions in mammalian cells. We have recently cloned the cDNAs encoding human and murine PARP-2 (62 kDa), which catalyzes the formation of ADP-ribose polymers in a DNA damage-dependent manner (6). Of the three other PARP homologues that have been cloned, two have been characterized as follows. (i) Tankyrase, a multi-functional protein of 142 kDa with regions of homology to ankyrins and to the PARP catalytic domain, binds to and negatively regulates TRF1, a factor involved in telomere maintenance (7); (ii) VPARP, the 193-kDa Vault particle PARP, which contains a BRCT domain and regions similar to the inter-alpha -trypsin inhibitor protein and a central region homologous to the catalytic domain of PARP-1. It binds to and ADP-ribosylates the 100-kDa major Vault protein (MVP) (8). The third, PARP-3, a 61-kDa homologue has also recently been identified but needs to be further characterized (9).

Despite the structural similarity of their catalytic domain, it is unclear whether all five PARP homologues serve similar or related functions in the cell (10). PARP-1 and PARP-2 are clearly DNA damage-dependent enzymes, suggesting a possible functional redundancy. However, a possible role for the latter in the same base excision repair pathway as PARP-1 (11) remains unknown.

To elucidate the role of PARP-2 in these various processes and as an essential step toward the development of an animal model, we cloned the mouse genomic PARP-2 sequence and compared the structure of its promoter and gene to that of PARP-1. To our surprise, we found that the 5' end of the PARP-2 gene lies within 113 and 152 base pairs of the 5' end of the gene encoding the RNase P RNA in mouse and human, respectively. The elements responsible for the activity of this bi-directional promoter (RNase P RNA/PARP-2) encoding a class III and a class II gene, respectively, were subsequently determined. Although these transcriptional elements are shared because of the compactness of the promoter region, comparison of the expression of both RNase P RNA and mPARP-2 mRNA in mouse showed that the regulation of the expression of the two genes is not coordinated.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genomic Library Screening and Sequence Analysis-- The PARP-2 gene was isolated from a mouse genomic library (kindly provided by J. M. Garnier, IGBMC, Illkirch, France) constructed from mouse embryonic stem cells (ES cells, strain SV129D). DNA was partially digested by MboI and inserted into the BamHI site of the lambda GEM12 vector. Screening of the library was performed using an 810-bp random-primed 32P-labeled cDNA probe corresponding to the 5'-most fragment of the mouse PARP-2 open reading frame extending from nucleotide 29 to nucleotide 838 (natural EcoRI site). Nylon membrane replicas of about 1×106 plaques were prehybridized at 67 °C for 2 h in 5× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), 1× Denhardt's solution, 2 mM EDTA, 0.1% SDS, and 0.1 mg/ml salmon sperm DNA. Hybridization was carried out at 67 °C overnight under the same conditions. Membranes were washed for 10 min in 2× SSC, 0.1% SDS at room temperature and then in 1× SSC, 0.1% SDS followed by 0.1× SSC, 0.1% SDS for 10-20 min at 65 °C and exposed to x-ray film at -80 °C overnight. Positive plaques were isolated and rescreened using the same probe. After three rounds of screening, the DNA of eight purified positive recombinant phages was isolated and digested by SacI. The fragment order was analyzed by Southern blotting with the library-screening probe and with synthetic oligonucleotide probes corresponding to the PARP-2 cDNA. SacI-generated DNA fragments of interest were subcloned into the pBluescript SK vector and sequenced from both directions by the chain termination method (12) using T3 and T7 primers or internal PARP-2-specific oligonucleotides. Homology searches were performed by comparing the nucleotide query sequence against a nucleotide sequence data base using the NCBI program BLASTN (13).

Genomic Sequence Analysis-- Sequence comparisons were performed using the collection of programs from the GCG package and from the European Molecular Biology Open Software Suite (EMBOSS, Sanger Center, UK). Potential transcriptional factor binding sites within the human and the mouse sequences were identified by searching against the TRANSFACT data base release 3.5 using the "tfscan" program (EMBOSS).

RNA Analysis; Primer Extension-- Poly(A)+ mRNA was purified from PARP-1+/+ and PARP-1-/- 3T3 cells using the messenger RNA isolation kit from Stratagene (La Jolla, CA). Total mouse tissue RNA was purchased from Ambion (Austin, TX). Transcription start sites on the mouse and human PARP-2 genes were mapped by reverse transcriptase primer extension essentially as described (14). A synthetic oligonucleotide (3'OH-ACCGCGGCGCCGCCGTCTCTAGTCCGA for mouse and 3'OH-ACCGCCGCGCCGCCGCTGCCTCGTGGCCG for the human) spanning the PARP-2-transcribed strand from positions +90 to +64 for the mouse and from positions +107 to +79 for the human mRNA was used as primer for the reverse transcriptase reaction. The reaction was carried out under standard conditions indicated by the enzyme supplier (Biolabs) but at two different temperatures, 10 min at 42 °C followed by a 10 min step at 50 °C. The same oligonucleotides were also used to sequence the mouse and human genomic regions cloned into a Bluescript vector (clones pBS-10G6 and pBS-hH1-P2, respectively). Reaction products were separated on an 8% polyacrylamide denaturating gel and autoradiographed overnight.

Recombinant Plasmid Construction-- A series of promoter deletion mutants of the mPARP-2 gene were generated spanning from -440 bp to -119 bp of the mouse transcription start site using PCR primers containing HindIII and BamHI restriction sites with clone 10G6 as template. All mutants shared the 3'-end primer (3'-AGCTTAGAATCTAAACCACAGACATcctagg-5'). The sequences of the 5'-end primer were as follows: pEGFP-RNAH1, 5'-aagcttCCAAGGATCTCGATCAAAAAGGAGGGC-3'; pEGFP-PromTATA, 5'-aagcttCCCACTTTTAGGGTGTAGACCGGCCGCCA-3'; pEGFP-PromTATA-Less, 5'-aagcttGGGTGTAGACCGGCCGCCACgaattcGGCTCGAAAGA-3'. DNA amplification was performed in 50-µl volumes containing 1× PCR buffer, MgCl2 (1.5 mM), dATP, dCTP, dGTP, and dTTP (0.2 mM each), DNA primers at 10 µM, 20 ng of clone 10G6 as template, and Taq polymerase (5 units, Promega, Madison, WI). The PCR products were first cloned into the TA-cloning vector pCR2.1. The resulting recombinant vectors were digested with HindIII and BamHI. The inserts generated were subcloned into the plasmid pEGFP-1, which contains the green fluorescent protein (GFP) gene (CLONTECH, Palo Alto, CA). To examine whether the mPARP-2 promoter allowed initiation of transcription in the reverse orientation, a DNA fragment generated by an EcoRI digestion of pCR2.1-PromTATA was subcloned into pEGFP-1 in the reverse orientation, giving pEGFP-PromTATAr. The Oct-1 binding site was mutated using PCR primers with the linearized pEGFP-PromTATA clone as template. Two PCR reactions were necessary to generate the mutant. The first reaction used a 5' primer that overlapped the Oct-1 binding site and replaced the Oct-1 sequence with the sequence of the XhoI restriction site; its sequence was as follows: 5'- TCCCACAAAGCACAGCGCGTAtctcgagcGTGCTCTATCCCAGGCTC-3'. The 3'-end primer was the following: 3'-TCGCCGGACACGCTGAACTTGTGGCCGT-5'. The amplification product was used as a 3'-end primer in a second reaction with the following 5'-end primer: 5'-GGGTTTCGCCACCTCTGACTTGAGCGTCGA-3'. The 0.52-kb PCR product was digested with HindIII and BamHI then subcloned into a pEGFP-1 reporter plasmid giving pEGFP-PromTATA-Delta Oct-1.

mPARP-2 Gene-H1 RNA Gene "Colinearity"-- To confirm that the PARP-2 and H1 RNA genes are localized on the same molecule of DNA, the region spanning from the H1 RNA gene to the first intron of the mPARP-2 gene in both mouse and human was amplified by PCR. To obtain the predicted 1121-bp DNA fragment in the mouse, primer m1 (5'-ACAGTGGGAGGGGGTTCATATCAT-3'), complementary to the mouse H1 RNA gene, and primer m2 (5'-AGACCACTTCATCATGGTCATCA-3'), complementary to the first intron of the mPARP-2 gene, were used with either the 10G6 clone or NIH/3T3 cell total DNA as template. In the human, the primers were h1 (5'-AATGGGCGGAGGAGAGTAGTCT-3') and h2 (5'-GGTCACAGTAGGTGGCATCGTT-3'). The PCR reaction, using HeLa total DNA as template, generated the expected 691-bp DNA fragment. The presence of both genes on the amplified fragment was confirmed by Southern blot analysis (15) using 5' 32P-labeled oligonucleotide probes specific to each gene: mH1 (5'-AGTGGGCGGAGGAAGCTCATCA-3') and mP2 (5'-CTCGCCTTCCAGAGCCTG-3') for the H1 RNA and mPARP-2 genes, respectively, in the mouse and hH1 (5'-GGTACCTCAACCTCAGCCATTGA-3') and hP2 (5'-TTCGAATTCCATGGCGGCGCG-3') for the H1 RNA and mPARP-2 genes, respectively, in the human.

Transfection and mPARP-2 Promoter Analysis-- HeLa and NIH/3T3 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5 and 10% fetal calf serum, respectively. Twenty hours after plating, the cells were transfected by calcium phosphate coprecipitation (16) using 3 µg of supercoiled recombinant DNA per 35-mm dish. The medium was changed after 8 h and at 24 h, and transfection efficiency was determined using inverted fluorescence microscopy (Zeiss Axiovert 135). Cells were cotransfected at a 1:10 ratio with the control reporter vector pDSRed1-N1 (17), which gave a red signal on fluorescence microscopy. In a second group, propidium iodide (10 µg/ml) was used to control for background fluorescence. Promoter analysis by flow cytometry was performed by recording green fluorescence intensity emitted by the transfected cells 36-48 h after transfection. Cells were harvested by trypsinization, resuspended in 1× PBS, and then placed in ice until analysis by flow cytometry (Becton Dickinson Co. FACScanTM flow cytometer). Data were acquired and analyzed using the program CellQuestTM. Variations in the GFP reporter gene expression were controlled by dividing the arithmetic mean of GFP expression by the equivalent measure for RFP expression in the same cells.

Preparation of Cell Extracts-- Whole cell extracts were prepared as previously described (18). Briefly, after trypsinization, NIH/3T3 cells were concentrated by centrifugation and washed twice with ice-cold PBS. After one additional wash in 10 mM KCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 10 mM Hepes, pH 7.9, the cells were resuspended in one packed cell volume of the same buffer and allowed to swell for 10 min. After swelling, the cells were disrupted by Dounce homogenization until most nuclei were broken. The homogenate was made 200 mM in KCl by the addition of 1 M KCl and further homogenized by 5 strokes of the Dounce homogenizer before centrifugation at 26,000 × g for 10 min. The resultant supernatant was dialyzed for 4 h against 100 volumes of 100 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20% glycerol, 20 mM Tris-HCl, pH 7.9. After dialysis, the homogenate was centrifuged at 10,000 × g for 15 min, and the resultant supernatant was divided into aliquots of 150 µl and stored at -80 °C.

Transcription Reactions in Vitro-- Transcription reactions were carried out in a final volume of 25 µl. The mixtures contained 15 µl of the whole cell extract, 5 mM MgCl2, 80 mM KCl, 0.5 mM dithiothreitol, 20 mM creatine phosphate, 0.5 mM each ATP, UTP, and CTP, 50 µM GTP, 5 µCi of [alpha -32P] GTP, 12 mM Tris-HCl, pH 7.9, and DNA templates as indicated in the figure legends. After a 1-h incubation at 30 °C, reaction mixtures were diluted to 250 µl in 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.1 M NaCl, 0.5% SDS, digested with 100 µg/ml proteinase K for 30 min at 42 °C, and extracted with phenol/chloroform followed by an ethanol precipitation. Reaction products were analyzed on M urea, 6% polyacrylamide gels. After electrophoresis, labeled transcripts were visualized by autoradiography.

In Situ Hybridization-- Serial sections of a 15-day-post-coïtum mouse (10 µm) were made using a standard cryostat, collected on gelatin-coated slides, and stored at -80 °C.

The murine PARP-2 probe corresponded to a cDNA fragment encoding residues 8-363. This fragment, cloned into pBluescript SK(+), was obtained by the screening of a 10-day-old mouse embryo cDNA library (6). The antisense mPARP-2 probe was produced by linearization with XbaI and transcription using the T7 promoter, whereas the sense probe was produced by linearization with XhoI and transcription using the T3 promoter. The murine RNase P RNA antisense probe was generated by an EcoRI run off T3 in vitro transcription of a 261-bp SmaI-Eco RI DNA fragment from the genomic clone 10G6 (see "Results") subcloned into pBluescript SK(+). The control sense RNase P RNA was produced by an SmaI run-off T7 in vitro transcription of the same construction. In situ hybridization was performed as described in Niederreither and Dollé (19). Exposure was for 1 week for RNase P RNA probe and 6 weeks for PARP-2 probe.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Organization of the Mouse PARP-2 Gene-- A mouse genomic library was screened with a PARP-2 cDNA probe that spans the coding region from amino acids 11 to 281, including a major part of the DNA binding domain and its putative nuclear location signal and nearly half of the catalytic domain. After hydrolysis by SacI, DNAs from eight clones were mapped by Southern blot analysis using the screening probe and oligonucleotides derived from the PARP-2 cDNA sequence. Two overlapping positive genomic clones, phages 5 (15.7 kb) and 10 (14 kb), were chosen as they span the whole PARP-2 gene (Fig. 1). SacI DNA fragments belonging to PARP-2, 10G6 (6 kb), 10G3 (3 kb) for phage 10 and 5G2.5 (2.5 kb), 5G5 (5 kb), 5G6.5 (6.5 kb) for phage 5 were subcloned into the pBluescript SK vector.



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Fig. 1.   Structure of the mouse PARP-2 gene and comparison with the mouse PARP-1 exon organization. The gene structure is shown with length in bp, as deduced from Southern blot analysis with PARP-2 cDNA oligonucleotide probes. Complete nucleotide sequence analysis (GenBankTM accession number AF191547) of the corresponding SacI DNA fragments isolated from two positive genomic clones, phage 10 and 5, are aligned below. PARP-2 exons (1-16) as well as RNase P RNA (H1 RNA, GenBankTM accession number L08802) are represented as closed boxes. Mouse PARP-1 protein and its exon splitting (21) have been aligned with the PARP-2 protein. NLS, nuclear location signal; DBD, DNA binding domain.

Sequence determination and comparison with the previously established cDNA sequence (6) allowed us to position the introns and exons and to set up the gene structure. As shown in Fig. 1, the PARP-2 gene (about 13 kb in length) comprises 16 exons, all of them shorter than 150 bp except exon 16, which is more than 260 bp long, and 15 introns with sizes varying from 80 bp (intron 10) to 3044 bp (intron 4) (Table I). The sequences at the exon/intron junctions were in very good agreement, 53-100% homology, with the consensus sequences (20) around both splice donor and acceptor sites (Table I).


                              
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Table I
Exon/intron organization of the mouse PARP-2 gene
The sequences at each exon/intron junction are aligned, and the length in bp of each exon and intron is indicated. The position of the boundary is shown with respect to the PARP-2 amino acid sequence reported below the nucleotide sequence (the number, in italics, corresponds to the last coded amino acid in exon n). Nucleotides identical to the consensus sequence (20) of splice donor (DS) and acceptor (AS) sites are in bold (100% identity) or are underlined (>53% identity).    

Organization of the PARP-2 Gene vis à vis the Protein Modules-- The PARP-2 protein has been postulated to be organized in three main modules based on their respective biochemical function (Fig. 1). The first module, spanning from amino acids 1-65, contains the DNA binding domain as well as the nuclear targeting sequence (6). The second module spans amino acids 65-198 and corresponds to domain E of PARP-1. The third domain (termed F in PARP-1) spans amino acids 199-559 and contains the catalytic site of the enzyme. The information we obtained on the genomic organization allowed a matching of these domains to specific groups of exons of the PARP-2 gene (Fig. 1). The DNA binding domain/nuclear location signal domain (amino acids 1-65) predicted to be responsible for the specificity of PARP-2 function is encoded by exons 1 and 2. Exons 3-7 code for domain E, whose function is still unknown. Finally, the catalytic domain (amino acids 198-559 corresponding to domain F in PARP-1) contains the evolutionarily conserved PARP signature and is encoded by exons 8-16. Only one of the intron-exon junctions in the catalytic domain was found conserved between the PARP-1 and PARP-2 genes (21, 22). This is the junction between exon 7 and exon 8 in PARP-2, which is identical to the junction between exon 13 and exon 14 in PARP-1. In both cases, the limit between domains E and F is delineated by the same amino acid. This exon/intron boundary is also the only one conserved in the PARP-3 gene and is located between exons 3 and 4 (data not shown). The absence of conservation of the other intron/exon boundaries at least in the PARP genes already described, strongly suggests that these genes must have evolved independently.

PARP-2 Gene and H1 RNA Gene Are Close Neighbors-- The surprising feature of the genomic structure of the PARP-2 gene was its extreme proximity to the RNase P RNA (H1 RNA in human) gene. To exclude any possible cloning artifact, we verified the relative position of the two genes in both mouse and human genomes by two PCR experiments and a Southern blot analysis. Four specific oligonucleotides complementary to the PARP-2 and RNase P RNA genes were designed to amplify a DNA fragment that would contain the entire exon 1 of the PARP-2 gene and almost the whole RNase P RNA gene in both mouse and human as shown in Fig. 2A. Both PCR products were of the expected sizes (1121 and 691 bp for the mouse and human, respectively) deduced from the genomic sequences shown on a 1.2% agarose gel (Fig. 2B). Southern blot analysis was then performed to demonstrate the presence of both genes within the same DNA molecule. Radiolabeled oligonucleotide probes specific to sequences located within the mouse and human PARP-2 and RNase P RNA genes (mP2, mH1, hP2, and hH1, respectively) were hybridized to the PCR DNA fragments. Fig. 2B shows that all four probes produced a strong signal, indicating the presence of the two genes within a single DNA fragment. This confirmed the gene structure described above as well as its conservation in both the mouse and human.



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Fig. 2.   PARP-2 and H1 RNA genes are located on the same genomic DNA fragment. A, schematic representation of the PCR and Southern blot strategies in the mouse and human. Relative positions of both the mouse and human primers (m1, m2 and h1, h2, respectively) and the oligonucleotide probes (mH1, mP2 and hH1, hP2, respectively) complementary to the H1 RNA and PARP-2 genes are shown. B, EtBr, ethidium bromide-stained 1.2% agarose gel of the amplified DNA fragments using mouse genomic DNA (lanes 1 and 3), clone 10G6 isolated from the mouse genomic library (lanes 2 and 4), and human genomic DNA isolated from HeLa cells (lanes 5 and 6) as templates. Southern autoradiograph representing identification of the mouse H1 RNA gene (lanes 1 and 2) and the PARP-2 gene (lanes 3 and 4) and the human H1 RNA gene (lane 5) and PARP-2 gene (lane 6) on a Southern blot of the gel shown above. The respective 5' 32P-labeled oligonucleotide probes are indicated.

Determination of the Transcription Start Site-- As a first step toward the delineation of the promoter region of the mouse PARP-2 gene, we determined the transcription start sites. Primer extension analyses were performed on poly (A)+ mRNA purified from PARP-1+/+ and PARP-1-/- 3T3 total RNA and on total RNA isolated from mouse ovary and testis tissue (Fig. 3). In all cases, primer extensions clearly indicated that the mPARP-2 cDNA was transcribed from a unique transcription start site, a cytosine at -63 bp from the initiation of translation (Fig. 3 and 4). Similar experiments were performed to determine the transcription start sites in the human PARP-2 gene. It was located close to the mouse start site, but -78 bp from the initiation of translation (data not shown) (Fig. 4). The position of the initiation region was close to the most 5'-extending mouse and human PARP-2 cDNA originally isolated (6). This confirmed the size of the protein we described (6) as opposed to the shorter form of PARP-2 identified by Berghammer et al. (23) and Johansson (9).



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Fig. 3.   Mapping of the mouse PARP-2 gene transcription start site. A, a diagram of the primer used for reverse transcriptase primer extension (RT) is presented (see "Experimental Procedures" for details). Coordinates were given a posteriori with respect to the +1 start site. B, reverse transcription was carried out using total mRNA isolated from various mouse cell types and tissues: wild type 3T3 (PARP-1+/+), PARP-1 knock out 3T3 (PARP-1 -/-), ovary, and testis. Sequence reactions were performed using the same primer on clone 10G6 as template and were run in parallel (lanes A, C, G, and T). The resulting ladders were used to position reverse transcriptase primer extension signals: relevant nucleotide sequences are given on the left with the initiation site indicated.



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Fig. 4.   Nucleotide sequence comparison of the mouse and human H1 RNA and PARP-2 genes and promoter region. PARP-2 cDNA nucleotide sequence is in small caps. H1 RNA nucleotide sequence is in small gray caps and underlined. The human sequence has been taken from Altman et al. (18) (accession number X16612). TATA, PSE, DSE/OCT1 boxes are highlighted. Consensus sequences for the transcription factor binding elements are indicated as solid gray bars. Sp1, ubiquitous zinc finger transcriptional factor. USF, a factor involved in development. E2F, involved in the regulation of cell cycle. Overexpression of this factor may cause neoplastic transformation and autoregulate its own promoter in a cell cycle-dependent manner; its expression is lost in senescent cells. CREB, is a strong activator, mediating gene activation in response to cyclic AMP after phosphorylation. SRF01 binds to serum response element (SRE). VBP01 appears to have a pivotal role in the estrogen-dependent regulation of the chicken vitellogenin gene, similar to DBP (rat). IK2, inhibitor of NF-kappa B, c-Rel, and v-Rel; dissociates NF-kappa B·DNA complexes. XBP binds to the X-box elements of class II MHC genes. OCT1, transcriptional activator in the polymerase II and in the polymerase III system. STAF, selenocysteine tRNA gene transcription-activating factor. Transcriptional activators Staf and Oct-1 play critical roles in the activation of small nuclear RNA and small nuclear RNA-type gene transcription. Transcription sites are indicated by arrows. The mouse and human intergenic region is 113 and 152 bp, respectively.

Determination of Promoter Architecture-- The comparison of the mouse and human promoter sequences between the PARP-2 and the RNase P RNA genes is shown in Fig. 4. In both cases, this region, which spans the 3'-end of the RNase P RNA gene to the first intron of the PARP-2 gene, has an identical organization. It displays a high level of sequence homology even in the nontranscribed 5' regions of both genes, which correspond to their promoter region. As was noticed above, however, the striking feature of this promoter region was the short distance between the two genes. The intergenic distance between the PARP-2 and RNase P RNA start sites is 113 bp for the mouse and 152 bp for the human. This suggests that this DNA region originally described by Altman et al. (18) as the promoter for the human RNase P RNA gene (H1 RNA) could also be used for the transcription of the PARP-2 gene. As for the transcription of most of the small RNAs (U6, 7SK and 7SL, MRP RNAs), the transcription of the H1 RNA is achieved in vitro by the RNA polymerase III (18). Different cis-acting transcriptional control elements residing within the 5'-flanking region were identified as necessary and sufficient for the transcription of the H1 RNA in vitro (24). The authors also noticed that the H1 RNA gene promoter was surprisingly complex. In addition to the TATA, PSE (proximal sequence element) and DSE (distal sequence element) homologs (Fig. 4), multiple sequence elements were required for efficient H1 RNA synthesis in vitro (24). This complexity, which may now be explained by the presence of the PARP-2 gene lying head to head with the H1 RNA gene, together with the short distance between the two genes strongly suggests that the promoter is bi-directional. Computer analysis identified potential transcription factor binding sites such as STAF, VBP01, E2F, Oct1, CREB (cAMP-response element-binding protein), XBP, SRF01, IK2, and CP2. These sites are likely candidates for coordinated regulation of the H1 RNA and PARP-2 genes.

Functional Analysis of the PARP-2 Promoter-- Bi-directional promoters, although common in prokaryotes and viruses, are seemingly rare among higher eukaryotes and usually involve proteins sharing very similar structural and functional characteristics. This study is the first to report a gene organization resulting from the close proximity of their transcription start sites, where a structural RNA, known to be transcribed by RNA polymerase III, and a protein whose transcription requires RNA polymerase II share the same promoter unit. To address this, a series of GFP reporter constructs were generated by inserting various PCR-generated fragments into the pEGFP-1 promoter-less vector (Fig. 5A). The promoter activities were assessed by transient transfection of these reporter constructs initially into HeLa cells and then into NIH/3T3 cells. The level of green fluorescence was directly monitored by flow cytometry. The promoter activity was determined by the ratio between the mean fluorescence for a specific promoter construction and the mean fluorescence measured for the promoter-less pEGFP-1 vector (Fig. 5B). The highest level of transcription was obtained with the construct pEGFP-promTATA, which contained the whole promoter region spanning the 5' sequence of the H1 RNA gene (position -119) to the mPARP-2 ATG (position +63). The promoter strength was 7.5-fold more potent than the vector with no promoter but was 45-fold less potent than that of the pCMV promoter. This result reflected the level of expression of the PARP-2 protein in the cell that was found to be low compared with that of PARP-1 (6). The presence of the complete H1 RNA gene in addition to the promoter region, as in the pEGFP-RNAH1 construct, reduced significantly the transcription in the PARP-2 direction with a 2.5-fold decrease compared with that of the basal promoter. To test if the promoter could function as a bi-directional promoter, the basal promoter construct was cloned in the antisense orientation to the GFP sequences (pEGFP-PromTATAr). A substantial fluorescence was measured that was almost twice as high in HeLa as in mouse NIH/3T3 cells. This indicated that the promoter was also able to initiate RNA polymerase II transcription in the H1 RNA gene direction. This was unexpected, since H1 RNA was shown to be transcribed by RNA polymerase III (18, 24). Control element sequences that match the consensus sequences of control elements for RNA polymerase II had been identified and were found to be required for H1 RNA transcription (18, 24). These include a TATA box control element sequence located at positions -30 (distance from H1 RNA start site) for the human or -29 for the mouse, a PSE located at position -68 for the human or -66 for the mouse, and a DSE located at position -97 for the human or -95 for the mouse. To test the hypothesis that some of these control elements could be required for the transcription of the PARP-2 gene, the sequence 5'-TATAA-3' located at -89 (from mPARP-2 start site) and the sequence 5'-ATTTGCAT-3' located at -25 were replaced by unrelated sequences, giving pEGFP-PromTATA-Less and pEGFP-PromTATA-Delta Oct-1 constructs, respectively. Both TATA-less and Oct-1-mutant promoters essentially abolished transcriptional activity in the direction of the PARP-2 gene, retaining a similar effect as for the transcription of the H1 RNA gene. We also tested the influence of some intronic sequences located within the first intron of the PARP-2 gene. Four plasmids were designed as shown in Fig. 5 (constructs e-h), all extending 231 bp further down from the normal PARP-2 start site. The constructs containing the H1 RNA gene retained low transcriptional activity in both HeLa and NIH/3T3 cells. No expression of the GFP reporter was noticed in the absence of the TATA sequence and when the plasmid contained only the intergenic region in addition to the intronic sequence. These results showed that the first intron did not contain any important transcriptional elements nor any other initiation sites as was suggested previously (23).



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Fig. 5.   Functional analysis of the mouse PARP-2 promoter. The mouse PARP-2 promoter sequences that have been fused to the GFP reporter sequences in the promoter-less pEGFP-1 vector and transfected in HeLa (black bars) and in NIH/3T3 (gray bars) are schematically depicted on the left, with numbers referring to PARP-2 coordinates and arrows oriented in the direction of transcription. 36-48 h after the transfection, the fluorescence of 20,000-50,000 transfected cells was measured by flow cytometry. The relative GFP fluorescence from four independent transfection experiments were determined and are plotted on the right, with S.D. The constructs are as follow: a, pEGFP-RNAH1; b, pEGFP-promTATA; c, pEGFP-promTATA-Less; d, pEGFP-promTATA-Delta Oct-1; i, pEGFP-promTATAr. FACS, fluorescence-activated cell sorter.

RNA Polymerase III Transcription of H1 RNA-- To ascertain the nature of the polymerase that transcribes the gene for H1 RNA, transcription reactions were carried out in vitro in the presence of increasing concentrations of alpha -amanitin. As an internal control, these reactions contained an authentic RNA polymerase III template corresponding to a gene for 5 S rRNA from Xenopus borealis. H1 RNA and 5 S rRNA synthesis was assayed in whole cell extracts (Fig. 6). Synthesis of both transcripts showed an identical sensitivity to inhibition by alpha -amanitin. That is, the transcription of both RNA molecules was not completely inhibited even at the higher concentration of alpha -amanitin. These results indicate that the H1 RNA gene is transcribed by RNA polymerase III, as was previously demonstrated by Altman and co-workers (18).



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Fig. 6.   Inhibition of transcription of the gene for H1 RNA by alpha -amanitin. 250 ng each of plasmid DNA (pEGFP-promTATA, panel A) or (pEGFP-RNAH1, panel B) (see Fig. 7) or plasmid DNA containing a gene for X. borealis 5 S rDNA (p5S) were incubated with a whole cell extract of NIH/3T3 cells in the presence of increasing concentrations of alpha -amanitin (panel B). Panel A: lane 1, control with no plasmid; lane 2, control transcription reaction with pEGFP-promTATA and p5S. Panel B: lanes 1-8, transcription reaction with pEGFP-RNAH1 and p5S and no addition and 1, 2, 4, 8, 16, 32, or 64 µg/ml alpha -amanitin, respectively.

Comparative Expression of PARP-2 and H1 RNA in Mouse-- To examine if the close proximity of the two genes influenced their relative expression, we compared the transcription by in situ hybridization during mouse embryogenesis and in adult mouse organs. Whereas PARP-2 is differentially expressed (Fig. 7A and data not shown), the H1 RNA is ubiquitously transcribed (Fig. 7B). The level of expression of the H1 RNA is considerably higher than for PARP-2 (exposure times of the photographic emulsion were 7 and 40 days, respectively). These results indicated that although the promoter region is shared by the two genes, their expression is independently regulated.



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Fig. 7.   Comparative in situ analysis of PARP-2 (A) and H1 RNA (B) expression levels in a newborn mouse. Note the strong and homogenous labeling pattern obtained with the H1 RNA probe (B), contrasted with the differential tissue distribution of PARP-2 transcripts (A). Autoradiography exposure times were 40 days for the PARP-2 probe and 7 days for the H1 RNA probe.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The PARP-2-H1 RNA Sequences Localize within a "Hot" Genomic Area-- Both the human and mouse genomic sequences encoding the PARP-2 proteins have been localized on a conserved region of chromosome 14 (6) found to be the only syntenic region of this chromosome. These sequences map within a locus (14q11.2 for human and 14C1 for mouse) that harbors a number of genes playing essential cellular functions (apoptosis, chromosome ends maintenance, immune system): the neural retina leucine zipper (NRL) gene (25, 26), the adenylate cyclase-4 (ADCY4) gene (27), the CCAAT/enhancer-binding protein (C/EBP), epsilon gene (28), the granzyme B and H genes (29, 30), the T-cell antigen receptor, alpha  polypeptide (TCRA) (31-33) and Delta  polypeptide genes (TCRD) (34), the telomerase-associated protein-1 (TEP1) gene (35), BCL2-like 2 (BCL2L2) (36), and the APEX nuclease (multi-functional DNA repair enzyme) gene (37). A number of chromosomal abnormalities within this portion of chromosome 14, mainly translocations, have been found in a variety of human germ cell tumors, adenocarcinoma, leukemia, and lymphoma. Specifically, an inversion of the segment 14q11.2-q32.2 occurs in T-cell chronic lymphatic leukemia and a t (14;14)(q11;q32) translocation occurs in T-cell malignancies of patients with ataxia telangiectasia (31, 32). The multiplicity of alterations in the q11.2-12 region of chromosome 14 suggests that this region is particularly recombinogenic. It will be of great interest to determine the molecular basis of other observed malignant transformation phenotypes and to examine whether they are linked to alterations of the proteins encoded within this portion of chromosome 14, particularly PARP-2.

The interesting feature of the mouse PARP-2 gene is the presence in the reverse orientation of the RNase P H1 RNA gene at very close proximity to the 5' end region of PARP-2. The distance between the two transcriptional start sites is only 113 bp. In the human, the two genes are similarly organized. The high degree of sequence homology underlines the importance of the transcriptional control elements present in the intergenic sequence for the expression of both the PARP-2 and H1 RNA genes. We found this promoter region to be bi-directional. It drives the expression of both the PARP-2 and H1 RNA genes through RNA polymerase II and RNA polymerase III transcription, respectively. In the past, the human H1 RNA promoter was thought to be surprisingly complex (18, 24). A comparison with other RNA polymerase III transcription units revealed a number of similarities to the MRP, 7SK, and U6 small nuclear small nuclear RNA genes (38-41). The promoters for these genes have been extensively studied and have been shown to require cis-acting elements that are remarkably similar to the elements characteristic of RNA polymerase II promoters (42-45). As for these genes, the TATA, PSE, and DSE/Oct-1 homologue boxes were described as necessary elements for H1 RNA synthesis in vitro (18, 24). In our experiments, the independent replacement of the TATA and DSE/Oct-1 sequences by unrelated sequences dramatically affected the transcription of the GFP reporter by RNA polymerase II. It was barely detected in these mutants. This result strongly suggests that these sequence elements are shared by factors required for both RNA polymerases II and III transcription. Hannon et al. (24) suggest that the population of trans-acting factors necessary for H1 RNA synthesis is more complex than that required for the synthesis of other RNA polymerase III-transcribed small RNAs. This unusual requirement can certainly be explained by the presence of the PARP-2 gene. This bi-directional promoter has to accommodate the ubiquitous synthesis of H1 RNA as well as the tissue-specific transcription of the PARP-2 gene (Fig. 7). H1 RNA transcription can interfere with the expression of PARP-2 by the occlusion of the promoter by RNA polymerase III. This will strongly limit the access of the promoter to the RNA polymerase II transcription machinery. The activity of the larger promoter fragment (-440 to +63) containing the H1 RNA gene plus the promoter region fused to the GFP reporter gene in the direction of the PARP-2 promoter fragment was consistently lower than in the smaller -119 to +63 fragment containing the promoter alone. This suggested that the expression of H1 RNA somehow resulted in a lower expression of PARP-2. This is confirmed by the in situ hybridization experiment (Fig. 7), which shows in the whole embryo a high level of expression of the H1 RNA and a low expression of PARP-2 mRNA. We cannot, however, exclude the possibility that under certain conditions (DNA damages), the expression of PARP-2 could dramatically increase. This may affect the level of translation by reducing the amount of mature tRNAs available as a consequence of reduced H1 RNA expression. It would be a means for the cell to exert regulation at the translational level.

Compared with the promoter region of mammalian PARP-1s, the regulatory region of PARP-2 is very different. PARP-1 does not share its promoter with any other gene, and its organization is typical of that of housekeeping genes. It lacks a TATA box, but has GC-rich sequences (46, 47) (69% GC content versus 48% for the intergenic of PARP-2) with CpG dinucleotides, which are putative methylation sites often involved in the tissue-specific regulation of gene expression. The cis-acting regulatory sequences also differ between the two promoters. Those of PARP-1 are typical of many housekeeping genes from DNA polymerase beta  to histones (46, 48). These differences strongly suggest that the expression of PARP-1 and PARP-2 is independently regulated. In situ experiments showed their expression in almost all the same tissues, although generally, PARP-2 was expressed at lower levels (data not shown). The presence of the two proteins in the same cells suggested that their roles may be complementary, both having specific nuclear targets and functions reinforcing the need for independent regulation.


    ACKNOWLEDGEMENTS

We thank Dr. Shanti Natarajan-Amé for critical reading of the manuscript and Prof. Philippe Carbon and Prof. Claude Kedinger for helpful discussions. We also thank Caroline Waltzinger from IGBMC, Illkirch, France for expert advice on FACS analysis and Prof. Pierre Chambon for continual support.


    FOOTNOTES

* This work was supported in part by the Association pour la Recherche Contre le Cancer, Electricité de France, Fondation pour la Recherche Médicale, and the Commissariat à l'Energie Atomique.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 GenBankTM/EMBL Data Bank with accession number(s) AF191547.

Dagger Recipient of a postdoctoral fellowship from the Fondation pour la Recherche Médicale.

To whom correspondence should be addressed. Tel.: 33 390 24 47 07; Fax: 33 390 24 46 86; E-mail: demurcia@esbs.u-strasbg.fr.

Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M007870200


    ABBREVIATIONS

The abbreviations used are: PARP, poly(ADP-ribose) polymerase; GFP, green fluorescent protein; bp, base pair(s); kb, kilobase pair(s); PCR, polymerase chain reaction.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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