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
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ABSTRACT |
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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.
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- 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.
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 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 Recombinant Plasmid Construction--
A series of promoter
deletion mutants of the mPARP-2 gene were generated spanning
from 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 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 [ 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
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.
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.
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).
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.
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 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 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 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.
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,
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 (
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
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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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).
/
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.
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-
Oct-1.
80 °C.
-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 8 M urea, 6% polyacrylamide
gels. After electrophoresis, labeled transcripts were visualized by autoradiography.
80 °C.
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ABSTRACT
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DISCUSSION
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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.
Exon/intron organization of the mouse PARP-2 gene
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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.
/
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- B,
c-Rel, and v-Rel; dissociates NF-
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.
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-
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- Oct-1; i, pEGFP-promTATAr.
FACS, fluorescence-activated cell sorter.
-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
-amanitin. That is, the transcription of both RNA molecules was not
completely inhibited even at the higher concentration of
-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 -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
-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
-amanitin,
respectively.
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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
polypeptide (TCRA) (31-33) and
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.
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.
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.
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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.
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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.
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.
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