Regulation of poly(ADP-ribose) polymerase-1 (PARP-1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP-1
© Zaniolo et al; licensee BioMed Central Ltd. 2007
Received: 30 April 2007
Accepted: 25 October 2007
Published: 25 October 2007
Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously demonstrated that transcription of the PARP-1 gene essentially rely on the opposite regulatory actions of two distinct transcription factors, Sp1 and NFI. In the present study, we examined whether suppression of PARP-1 expression in embryonic fibroblasts derived from PARP-1 knockout mice (PARP-1-/-) might alter the expression and/or DNA binding properties of Sp1 and NFI. We also explored the possibility that Sp1 or NFI (or both) may represent target proteins of PARP-1 activity.
Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells. Co-immunoprecipitation assays revealed that PARP-1 physically interacts with Sp1 in a DNA-independent manner, but neither with Sp3 nor NFI, in PARP-1+/+ cells. In addition, in vitro PARP assays indicated that PARP-1 could catalyze the addition of polymer of ADP-ribose to Sp1, which also translated into a reduction of Sp1 binding to its consensus DNA target site. Transfection of the PARP-1 promoter into both PARP-1+/+ and PARP-1-/- cells revealed that the lack of PARP-1 expression in PARP-1-/- cells also results in a strong increase in PARP-1 promoter activity. This influence of PARP-1 was found to rely on the presence of the Sp1 sites present on the basal PARP-1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP-1-/- cells. Subjecting PARP-1+/+ cells to an oxidative challenge with hydrogen peroxide to increase PARP-1 activity translated into a dramatic reduction in the DNA binding properties of Sp1. However, its suppression by the inhibitor PJ34 improved DNA binding of Sp1 and led to a dramatic increase in PARP-1 promoter function.
Our results therefore recognized Sp1 as a target protein of PARP-1 activity, the addition of polymer of ADP-ribose to this transcription factor restricting its positive regulatory influence on gene transcription.
Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly evolutionary preserved, multifunctional nuclear enzyme that catalyzes the addition of ADP-ribose (ADPr) units from nicotinamide adenine dinucleotide (NAD+) on a large variety of nuclear proteins and consequently impinges on many of the major nuclear functions (reviewed in ). Gene inactivation studies have revealed and/or confirmed up to nine biological functions for PARP-1 . These include both DNA repair and maintenance of genomic integrity as well as regulation of telomerase activity [3, 4] (also reviewed in ). PARP-1 also regulates the expression of various proteins at the transcriptional level (reviewed in ) and is also involved in DNA replication as well as cell differentiation [6–8]. In addition, polymer of ADP ribose (PAR) has been recently identified as an emergency source of energy used by the base-excision machinery to synthesize ATP [9, 10]. PAR may also serve as a signal to induce cell death . Finally, PARP-1 may contribute to the regulation of cytoskeletal organization and in the post-transcriptional modification of nuclear proteins like histones and transcription factors (reviewed in [2, 12]).
PARP-1 plays vital functions in gene transcription as it can influence the state of chromatin remodeling through the catalytic addition of PAR to the core histones [13, 14]. Besides, PARP-1, through its double Zn-finger DNA binding domain, can interact with target regulatory elements present in the promoter of many genes, including the MCAT 1 , Pax-6 , MHC II , and the CXCL1  genes. By exploiting both chromatin cross-linking and immunoprecipitation assays, Soldatenkov and coworkers elegantly demonstrated that PARP-1 could also bind to secondary hairpin-like structures present in the 5'-flanking region of the human PARP-1 promoter . Although no prototypical sequence that could be recognized as a target by the PARP-1 DBD has been identified yet, Pion and collaborators have however demonstrated the ability of the protein to bind to both 5'- and 3'-recessed ends on double-stranded DNA, as well as to palindromic-like structures often present in DNA . Lastly, PARP-1 can also alter gene transcription through its ability to poly(ADP-ribosyl)ate several transcription factors, such as YY1 , NFKB , TFIIF , Oct-1 , B-MYB  and AP-2 , thereby preventing their binding with their specific promoter target sites .
The studies conducted over the last few decades have been mostly dedicated to the study of the many biological and cellular functions of PARP-1. Unfortunately, not so many studies have explored the molecular mechanisms that regulate the transcription of PARP-1 gene expression. Unlike its enzymatic activity, PARP-1 gene transcription is not activated by DNA strand breaks [28, 29]. On the other hand, PARP-1 gene expression appears related to cell proliferation rather than DNA synthesis as PARP-1 mRNA has been shown to be more abundant in the G1 phase of the cell cycle [30–32]. To date, the PARP-1 promoter has been cloned from three different mammalian species: human , rat  and mouse . All three mammalian basal promoters share structural similarities typical of housekeeping genes in that they lack a functional consensus TATA box, possess a high content of GC-residues, and bear a consensus initiator sequence (Inr) that overlaps the transcription initiation site. The human promoter has been shown to contain binding sites for the transcription factors Sp1, AP-2 , YY1 , and Ets , whereas the mouse promoter was recently shown to be down-regulated by a complex of adenovirus E1A protein and pRb . Studies that we conducted on the rat PARP-1 (rPARP-1) proximal promoter have shown that its activity is primarily, but not entirely dependent on the recognition of five GC-rich binding sites (F1, F2, F3, F4 and US-1) by the positive transcription factors Sp1 and Sp3 [37, 38]. Other transcription factors, such as those that belong to the Nuclear Factor I (NFI) family of transcription factors , were also found to bind to nearby target sites to alter the activity directed by the PARP-1 gene promoter [39–41]. Sp1 and Sp3, two members from a Zn-finger family of transcription factors that presently comprises nine proteins (Sp1 to Sp9) , often compete with each other for the recognition of their common GC-rich target sites [43, 44]. However, Sp1 is a transcriptional activator, while Sp3 can, according to the context, function either as a repressor or an activator of gene transcription [43, 45]. The NFI family is composed of four members encoded by distinct genes, NFI-A, -B, -C, and -X , producing distinct protein products, which can form homo- or heterodimers . In addition, all four NFI mRNAs can be differentially spliced to yield a large number of NFI isoforms with subtle differences in their transactivation properties [48, 49]. NFI has been reported to repress the activity directed by the rPARP-1 promoter [39, 40]. However, as NFI was found to be transcriptionally inert by itself as it possesses no intrinsic activity in the regulation of the rPARP-1 promoter, its negative influence was suggested to result from the fact that it competes with Sp1 for the availability of a promoter composite element that bears overlapping target sites for both these transcription factors . Consequently, by exploiting either the synergistic or antagonistic effects of the transcription factors they bind to respectively over-activate or down-regulate transcription, the combination of regulatory elements that constitute the PARP-1 promoter provides an efficient way of fine-tuning its expression in different cellular contexts.
As with many other nuclear proteins, both Sp1 and NFI can be subjected to post-translational modifications through mechanisms such as phosphorylation and/or glycosylation [50, 51]. However, neither of these transcription factors, which are both required to ensure proper transcription of the PARP-1 gene, has been shown to be also subjected to poly(ADP-ribosyl)ation by PARP-1. In the present study, we examined both the expression and DNA binding properties of Sp1 and NFI in embryonic fibroblasts cultured from normal mice that express the intact, wild-type PARP-1 protein (PARP-1+/+), or from PARP-1 knockout mice (PARP-1-/-) that are devoided of any PARP-1 enzymatic activity . We demonstrated that PARP-1 physically associates, through protein-protein interaction, with Sp1 but not Sp3 or NFI in immunoprecipitation assays. Sp1 was also found to be a target of PARP-1 as addition of PAR to this transcription factor could be demonstrated. Preventing poly(ADP-ribosyl)ation of Sp1 in the PARP-1 deficient knockout cells also resulted in a substantial increase in the activity directed by rat PARP-1 promoter, a clear indication that addition of PAR to Sp1 reduces its transactivation properties in vivo.
Expression and DNA binding of Sp1, Sp3 and NFI in PARP-1+/+ and PARP-1-/- cells
The identity of the proteins that yielded the shifted complexes using the Sp1 labeled probe was further investigated by supershift analyses in EMSAs using antibodies directed against either Sp1 and Sp3, two closely related transcription factors from the same family . As shown on Figure 2D, the Sp1 Ab strongly reduced the migration of the complex with the lowest electrophoretic mobility but had no influence on the weaker, faster migrating complex. On the other hand, the antibody against Sp3 only slightly decreased the electrophoretic mobility of the slow complex but entirely competed the fast migrating one. Incubation of both the Sp1 and Sp3 Abs together entirely supershifted both complexes, using the nuclear extracts from both the PARP-1+/+ and PARP-1-/- cell lines. Both antibodies yielded super-shifted complexes (SSC) corresponding to the signal yielded by the Ab-Sp1(Sp3)-DNA complex. These results indicate that the slow migrating complex is made up of both Sp1 and Sp3 bound to the labeled probe (primarily Sp1) whereas the faster migrating complex is entirely made up of Sp3. A similar experiment was also conducted on the DNA-protein complex yielded by the incubation of both the PARP-1+/+ and PARP-1-/- nuclear extracts with the NFI probe. As presented on Figure 2D, addition of the NFI Ab reduced the electrophoretic mobility of the NFI complex from both the PARP-1+/+ and PARP-1-/- protein extracts and yielded a new super-shifted complex resulting from the recognition of the NFI-DNA complex by the NFI Ab. We therefore conclude that expression and DNA binding of both Sp1 and NFI is severely reduced in the PARP-1 knockout (PARP-1-/-) cells.
PARP-1 physically interacts with Sp1
To further validate the results from the Sp1 immunoprecipitation, the reverse experiment was conducted by first immunoprecipitating the PARP-1 associated proteins from total protein extracts prepared from PARP-1+/+ and PARP-1-/- cells using the F1–23 Ab against PARP-1. We selected this Ab primarily because it is much more efficient in immunoprecipitating PARP-1 than the C-2-10 Ab. The transcription factors Sp1, Sp3 and NFI could be detected easily by Western blot in the total extracts from both PARP-1+/+ and PARP-1-/- cells prior to immunoprecipitation of PARP-1 (T.E. in Figure 4B). Immunoprecipitation of PARP-1 with the F1–23 Ab and further Western blotting of the precipitated proteins revealed clearly that the F1–23 Ab was efficient in immunoprecipitating PARP-1 in the extract from PARP-1+/+ but not from PARP-1-/- cells. In addition, Sp1 could also be revealed in the immunoprecipitate using the sc-59 Ab. On the other hand, the Abs against Sp3 and NFI could not detect any of these transcription factors in the PARP-1 immunoprecipitate. We therefore conclude that Sp1 but not Sp3 nor NFI associate physically with PARP-1 in vitro through protein-protein interactions.
PARP-1 enzymatic activity is not required for its interaction with Sp1
Sp1 is a target of PARP-1 enzymatic activity
There are many potential hypotheses that may explain why expression of both endogenous Sp1 and PARP-1 does not change upon incubation with PJ34. However, and as the cells treated with PJ34 were harvested only 18 hours later, we initially thought they might not have had sufficient time to alter their pattern of protein expression. We therefore conducted a new set of experiments but selected primary cultures of human skin keratinocytes (HSKs) as a cellular model for PARP-1 expression rather than PARP-1+/+ cells. HSKs were first exposed to H2O2 in order to trigger activation of endogenous PARP-1 in these cells, and then harvested at various periods of times (4-, 24- and 72 hours) for further analysis of Sp1 DNA binding and both Sp1 and PARP-1 expression at the protein level. As an additional control, cells triggered with H2O2 were also added PJ34 at each selected time-point prior to harvesting. Neither H2O2 nor H2O2+PJ34 had any significant influence on the DNA binding properties of Sp1 at 4 h of treatment (Figure 7C; compare lane 1 with lane 2), which is also supported by the weak influence of these reagents on both Sp1 and PARP-1 protein expression in Western blot (Figure 7D). However, challenging PARP-1 activity through exposure of HSKs to H2O2 not only reduced the binding capacity of Sp1 (and to some extend, Sp3 as well) in these cells at 24 and 72 h (Figure 7C) but also translated into a reduction in the expression of both PARP-1 and Sp1 proteins (Figure 7D). Most interestingly, the H2O2-dependent reduction in the DNA binding of both Sp1 and Sp3 was not solely reverted by the further addition of the PARP-1 inhibitor PJ34 but was even considerably improved at 72 h (Figure 7C; compare lane 9 with lane 8). This dramatic increase in Sp1 DNA binding also translated in an increased expression of Sp1 at the protein level, which nearly doubled upon addition of the PJ34 inhibitor (ratio of the Sp1/β-actin signal of 0.111 with H2O2 and 0.206 with H2O2+PJ34, as revealed by densitometric analysis of each protein band). We therefore conclude that PARP-1-dependent addition of PAR to Sp1 reduces its DNA binding properties and thereby alter the efficiency with which it can transcribe its target genes, which comprise the PARP-1 gene besides from the Sp1 gene itself.
Influence of poly(ADP-ribosyl)ation on the activity directed by the rPARP-1 promoter
As the trans-activating properties of the PAR-free Sp1 is increased in PARP-1-/- cells, we then wished to determined whether the transcriptional activity directed by other gene's promoter reported to be under the regulatory influence of this transcription factor would also be similarly affected by the lack of PARP-1 activity. We recently reported that transcription of the human α5 integrin subunit gene was under the positive regulatory influence of both a proximal and a distal Sp1 site in primary cultures of rabbit corneal epithelial cells [57, 58]. We therefore used a recombinant construct that bear the CAT reporter gene fused to a truncated version of the α5 promoter that contain only the proximal Sp1 site (-92α5CAT) for conducting this experiment. In addition, and as the GC-rich promoter from the human α6 integrin subunit gene was also reported to be positively regulated by Sp1 [59, 60], a recombinant construct that has the CAT gene fused to the basal promoter from the α6 gene (α6–84) was also used in these assays. The -92α5CAT and α6–84 constructs were therefore transfected into both the PARP-1-/- and PARP-1+/+ cell lines and CAT activity determined and normalized. As shown on Figure 8B, transfection of the PARP-1 promoter-bearing construct pCR3 yielded a CAT activity that was more than 4-times more elevated in PARP-1-/- than in PARP-1+/+ cells (which is expressed as the ratio of the activity measured in PARP-1-/- over that from PARP-1+/+ cells). Interestingly, transfection of both the α5 and α6 promoter constructs also yielded increased CAT activities in PARP-1-/- cells (2.4- and 2.6-fold increases, respectively), indicating that the altered regulatory influence resulting from the lack of PAR addition to Sp1 in PARP-1-/- cells is not merely restricted to the PARP-1 promoter but also affect transcription of other Sp1-responsive genes as well.
The ability of the PARP-1 protein to interact with a variety of nuclear-located transcription factors and thereby alter their regulatory function toward the target genes that they regulate is certainly among the most important of the many functions PARP-1 may play as it will ultimately alter the pattern of genes expressed by any given cell. Although an increasing number of such transcription factors are being reported every year, yet only a few of them have been shown to be post-translationally modified by PARP-1. Here we show that PARP-1 physically associates with the positive transcription factor Sp1 and reduces its trans-activating properties by the catalytic addition of PAR to this protein. We demonstrate that such a post-translational alteration of Sp1 not only reduces the transcription of the PARP-1 gene itself (as the lack of PARP-1 activity in PARP-1-/- cells also translates into a more than 4-fold increase in PARP-1 promoter function, a result consistent with those reported by Soldatenkov et al. ), but also that of other Sp1-responsive genes, for instance those encoding the human integrin subunits α5 and α6. Based on the fact that the average branching frequency of the PAR is approximately one branch per linear section of 20–50 units of ADPr [61–63], and that ADPr units have a molecular mass of 577 Daltons, we can assume that PAR branching will translate in an increase of at least 11.5 kDa in the molecular mass of the acceptor protein. The unchanged apparent electrophoretic mobility of Sp1 in Western blot suggests that branching of ADPr units is however unlikely to occur. This is consistent with poly(ADP-ribosyl)ation of other transcription factors, namely RAP30/RAP74 subunits of TFIIF . An analysis of the 24 potential poly(ADP-ribosyl)ation sites (glutamic acids residues)in Sp1 (Accession no NP_612482) reveals that 8 of them are localized within the first 140 amino acids (N-terminal) whereas 12 are localized at the C-terminal end of the protein. Most of the sites are in hydrophilic portions of the protein (20 out of 24) and therefore available to accept PAR. The two last sites at position E752 and E757 are in an hydrophobic portion and might not be readily modified by PAR. Modification by PAR anywhere on the protein may induce its desorption from DNA. However, two potential sites (E672 and E690) are localized within the second and third Zn finger of Sp1, respectively. The specific targeting of these sites may be critical in negatively regulating the function of Sp1 on specific DNA sequence.
Sp1 modification by PAR alters the expression of the PARP-1 gene because of the presence of at least five distinct target sites for this transcription factor in the PARP-1 gene promoter . Besides its action on Sp1, PARP-1 has also been reported to regulate the transcription of its own gene through other regulatory mechanisms that are most likely related to the presence of secondary structures in DNA. Indeed, it has been shown that the DBD of PARP-1 can repress PARP-1 gene transcription by interacting directly with hairpin-like structures present in the promoter of the human PARP-1 gene . Although these results may appear contradictory at first sight to those we report in this study, yet the possibility remains that the PARP-1 truncated version they used might have caused repression of the PARP-1 promoter through the recruitment of other transcription factors rather than through a direct action of PARP-1 on Sp1. For instance, PARP-1 has been reported to physically interact with the transcription factors TEF-1, B-MYB and AP-2 [15, 16, 25, 26, 64]. Interestingly, all three transcription factors have been shown to function not only as activators but also as repressors of gene transcription [65–69]. Besides, the two most likely hairpin-like structures in the human PARP-1 promoter have been shown to be present at quite a distance from the Sp1 target sites-bearing basal promoter (from nt -325 to -290 and -418 to -403). It is also worth mentioning that mutation of all three Sp1 sites contained in the rPARP-1 basal promoter entirely suppressed the induction of PARP-1 promoter activity in PARP-1-/- cells, a clear evidence that the PARP-1 negative influence on its own promoter-driven transcription is entirely dependent on altering Sp1 binding at the rPARP-1 promoter.
Addition of PAR to transcription factors can either promote or reduce their affinity toward their corresponding sites in DNA. It has been shown that the DNA binding properties of a few transcription factors, notably YY-1, NFkB, CREB, the TFIID subunit TBP, and presumably Sp1, was severely reduced in the presence of NAD+ . The pattern of migration that they obtained for Sp1 on the EMSA was, however, not typical of that normally observed for this transcription factor and might have resulted either from protein degradation of the crude nuclear extracts used by these authors or from the fact that a transcription factor other than Sp1 has bound to the GC-rich labeled probe they used for conducting their EMSA. Sp1 belongs to a large family of transcription factors, the Sp/KLF family, which comprises 25 proteins that all possess the ability to bind GC- or GT-rich target sites in DNA as all of them share three similar Zn-fingers as their DNA binding domain . As no supershift experiments using an anti-Sp1 antibody were conducted in the study by Oei and coworkers, care must be taken as to whether Sp1 indeed was the transcription factor from the HeLa extract that actually bound their labeled probe in EMSA as the shifted band might have resulted from the binding of another member from the Sp/KLF family to the GC-rich labeled probe they used.
The experiments that we conducted either in vitro, using purified Sp1, or in vivo, by inducing the PARP-1 activity with the oxidative reagent H2O2, provided evidence that addition of PAR to Sp1 not only lowered its positive regulatory influence on gene transcription but also its capacity to physically interact with its high affinity target sites in DNA. This exposition of cultured cells (for instance HSKs) to H2O2 also translated in a reduced expression of both PARP-1 and Sp1 (as Sp1 was reported to regulate the transcription of its corresponding gene through the presence of multiple, functional GC-rich target sites [71, 72]) at the protein level, a process that could be reverted in a time-dependent manner by the PARP inhibitor PJ34 (Fig. 7). It is important to note that PJ34 has a broad range, and therefore inhibits the activity of not only PARP-1 but other PARPs as well. This would include PARP-2, a known DNA-dependent PARP involved in DNA damage response and repair [73, 74]. However, because PARP-1 accounts for more than 90% of cellular poly(ADP-ribosyl)ation , it makes the contribution of PARP-2, or other PARPs, marginal on modification of Sp1. Moreover, the use of PARP-1-/- cells strongly support the in vivo role of PARP-1 in the modulation of Sp1 activity. That no reduction, but rather an increase in the expression of Sp1 is seen in PARP-1+/+ cells (Figs. 1 and 2) is rather paradoxical with the results from the transfection experiments. Yet, the lack of any PARP-1 influence when all three Sp1 sites from the PARP-1 promoter-bearing construct PCR3 are mutated is a clear evidence that the primary action of PARP-1 in wild-type PARP-1+/+ cells is directed toward this transcription factor. As an interesting hypothesis, we suggest that in PARP-1 expressing cells, most, if not all Sp1 proteins has been added PAR to a certain level, which would represent a remarkable mean for the cell to restrict the otherwise very powerful trans-activating properties of Sp1 on gene transcription. Although the basal level of Sp1 expression is decreased in PARP-1-/- knockout cells whereas that of other transcription factors, such as Sp3, E2F1 and STAT-1, remains unaffected, it would however be free of added PAR and would thus become a much more potent activator of gene expression. Besides reducing its trans-activating properties, addition of PAR to Sp1 may also serve other cellular functions. Sp1 has been recently reported as particularly sensitive to protein degradation in quiescent, post-confluent, but not in actively growing primary cultures of rabbit corneal epithelial cells (RCECs) . Monitoring the steady state level of Sp1 in cells grown in the presence of PARP-1 inhibitors such as 3-AB or PJ34 in cells grown at a high cell density might prove particularly informative considering the recent demonstration that PARP-1 contributes to the activity of the proteasome in drug-induced, oxydatively damaged nuclear proteins [76–78]. Abnormally elevated expression and DNA binding of Sp1, as for many of the Sp/KLF family members to which belong Sp1 , has been recognized as a valuable prognostic marker in both gastric [80–82] and colorectal cancers . PARP-1 mediated addition of PAR to Sp1 can now be viewed as one out of a few other post-translational modifications, such as glycosylation and phosphorylation, that the cell may use to modulate the positive regulatory influence of Sp1 and thereby contribute to prevent any given cell to progress toward anchorage-independent, unregulated cell proliferation, the hallmark of all cancer cells.
Besides Sp1, our results also demonstrated a reduced expression of the transcription factors AP-1 and NFI in PARP-1-/- knockout cells relative to wild type in PARP-1+/+ cells, whereas that of others, such as Sp3, E2F1 and STAT-1 remained unaffected by the lack of PARP-1 activity. That AP-1 expression and DNA binding is strongly abrogated in PARP-1 deficient cells is a well documented fact [86–90]. Indeed, the pharmacological inhibition of PARP-1 activity in 3,4-dihydro-5-[4-(1-piperinidinyl)butoxyl]-1(2H)-isoquinoline (DPQ)-treated mice entirely abolished the TPA-dependent activation and DNA binding properties of AP-1 . Inhibition of PARP-1 activity in rats treated with nicotinamide was found to prevent N-methyl-N-nitrosourea-induced photoreceptor apoptosis through a reduction in the expression of the AP-1 subunit c-jun, a process that was postulated to depend on the inhibition of the JNK/AP-1 signalization pathway . PARP-1 activity was recently shown to be required for c-jun phosphorylation, a process that modulate the stability of AP-1 and determines its DNA binding efficiency . DNA binding of NFI was found to be strongly abrogated in the PARP-1-/- knockout cells. These alterations in the DNA binding properties of NFI also correlated with important changes in the pattern of NFI protein electrophoretic mobility on SDS gels that might result either from the expression of distinct NFI isoforms, or from post-translational modifications of NFI that may differ between the wild-type PARP-1+/+ and PARP-1-/- knockout cells. To our knowledge, no study has ever reported such a suppression of the NFI DNA binding properties in PARP-1-/- knockout cells. We previously demonstrated that NFI has a negative influence on the activity directed by the rPARP-1 promoter but not because it possesses any intrinsic repressor function but rather because it competes with Sp1 for the availability of a promoter composite element that bears overlapping target sites for both these transcription factors [39, 40]. Interestingly, members from the NFI family have been reported to be as efficient as repressors [40, 91, 92] than activators [93, 94] of gene transcription. Besides, NFI sites have often been shown to be located close to, or overlapping with nearby Sp1 sites. Indeed, NFI was reported to interact with and antagonize Sp1, which result in the down-regulation of the platelet-derived growth factor (PDGF)-A gene expression . We recently reported that a similar interference of Sp1 action by NFI might also account for the transcriptional repression of the p21 genes . The physiological significance of NFI suppression in the PARP-1-/- knockout cells has yet to be precisely determined, although one would predict that its suppression would tend to favor the positive influence of the potentiated action of Sp1 that results from its lack of PAR addition.
PARP-1 clearly contributes in a positive fashion to initiation of gene transcription by inducing local relaxation of the otherwise condensed chromatin by the attachment of PAR to the histones H1, H2A and H2B (reviewed in ). The recent demonstration that PARP-1 is closely associated to the enzyme topoisomerase IIβ (TopoIIβ ) further stressed the contribution of PARP-1 in the initiation of gene transcription as TopoIIβ, through its ability to create double-strand breaks in DNA, also provides a mean to activate PARP-1 enzymatic activity as well. Our results demonstrate that besides its positive action on gene transcription, PARP-1 may as well contribute to suppression of gene expression by its ability to add PAR to the strong transcriptional activator Sp1, thereby establishing a dual regulatory function (activation and repression) for this protein in gene expression.
Plasmids and oligonucleotides
The plasmid pCR3 as well as the pCR3 derivative plasmid that bear mutations in the three most proximal Sp1 binding sites (F2, F3 and F4; PCR3/F2F3F4m), have both been previously described . The recombinant plasmids α6–84 which bear the chloramphenicol acetyltransferase (CAT) reporter gene fused to a DNA fragment covering the human α6 gene promoter sequence from positions -84 to +76 relative to the α6 mRNA start site was constructed as follow: The pGEM-3Z f(+) AvaI/α6 plasmid that bears the human α6 gene promoter from position -887 to +76 relative to the α6 mRNA start site (and generously provided by Schei Kitazawa, Kobe University School of Medicine, Kusunoki-Cho, Chuo-Ku, Kobe) was linearized at its unique KpnI site (5'-end) and treated with the nuclease Bal31 before being blunted. The 5'-digested α6 promoter fragments generated were then digested with HindIII (3'-end at α6 position +76) before being ligated into the unique SmaI/HindIII sites of the plasmid pGL3 to derive the pGL3/α6–590 vector. The pGL3/α6–590 construct was then digested with NheI and blunt-ended with Klenow (which then preserved the α6 -590 5'-end), before being second-digested with XbaI (which preserved the α6 +76 3'-end). The resulting α6 promoter-bearing DNA fragment was then ligated upstream the CAT reporter gene from the pCATBasic vector (Promega) that has been first digested with Pst I (5'-end), blunt-ended with Klenow and second digested with XbaI (3'-end) prior to its dephosphorylation with alkalin phosphatase (to yield pCATBasic/α6–590). Removal of the α6 promoter fragment from the common SphI site (located upstream from α6 promoter position -590 in the multiple cloning site of the parental plasmid pCATBasic) to the internal restriction site SacII (at α6 position -84) followed by blunt-ending with Klenow and ligation with T4 ligase, yielded the plasmid α6–84. The PXGH5 plasmid is a kind gift of Dr. David D. Moore (Department of Molecular and Cell Biology, Baylor College of medicine, Houston, TX, USA). The double-stranded oligonucleotides bearing the high affinity binding site for the transcription factors Sp1 (5'-GATCATATCTGCGGGGCGGGGCAGACACAG-3') , Stat-1 (5'-AAGGCGGAGGTTTCCGGGAAAGCAGCACC-3') , AP-1 (5'-GATCCCCGCGTTGAGTCATTCGCCTC-3') , E2F1 (5'-CAGAGCCGGCGGGAAAGGTGCGGGCGGTGC-3')  and NFI (5'-GATCTTATTTTGGATTGAAGCCAATATGAG-3')  were chemically synthesized using a Biosearch 8700 apparatus (Millipore, Bedford, MA, USA).
Cell culture and media
The embryonic fibroblast cell lines derived from both normal (PARP-1+/+) and PARP-1 knockout mice (PARP-1-/-)  were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and gentamycin (20 μg/mL). Human skin keratinocytes (HSKs) were isolated from a normal adult skin specimen (26 year-old donor) removed during reductive breast surgery and grown on a feeder layer of irradiated mouse Swiss 3T3 fibroblasts in complete keratinocyte medium as described . Cells were maintained at 37°C, under a 5% CO2 controlled atmosphere in a humidified incubator. When indicated, both PARP-1+/+ cells and HSKs were grown in the presence of either 300 μM hydrogen peroxide (H2O2) for 10 min, and/or 1 μM PARP-1 inhibitor PJ34 for either 24 h (PARP-1+/+ cells) or for 4-, 24- and 72 h (HSKs); cells were also re-challenged (1 h prior to harvesting for PARP-1+/+cells, or every 24 h for HSKs) before they were harvested for the preparation of the nuclear extracts.
SDS-PAGE and Western Blot
The protein concentration from the nuclear extracts prepared from PARP-1+/+ and PARP-1-/- cells was determined by the Bradford procedure and precisely validated through Coomassie blue staining on 10% SDS-polyacrylamide gels (a typical example can be seen on Figure 3B). Either 20 μg nuclear proteins was added to 1 volume of sample buffer (6 M urea, 63 mM Tris (pH6.8), 10% (v/v) glycerol, 1% SDS, 0,00125% (w/v) bromophenol blue, 300 mM β-mercaptoethanol) and then size-fractionated on a 10% SDS-polyacrylamide minigel before being transferred onto a nitrocellulose filter. The blot was then washed in TS (150 mM NaCl, 10 mM Tris-HCl pH7.4) and TSM buffers (TS buffer with 5%milk and 0,1% Tween 20) as described previously . The membrane was further incubated for 1 h at 22°C with a 1:5000 dilution (except for the PARP-1 Ab (C-2-10) that has been used at a 1/10 000 dilution) of the following primary antibodies: rabbit polyclonal antibodies directed against the transcription factors Sp1 (sc-59; Santa Cruz Biotechnology, Inc. Santa Cruz, CA), Sp3 (sc-644; Santa Cruz Biotechnology), NFI (Santa Cruz Biotechnology, Inc.), c-jun (sc-44x; Santa Cruz Biotechnology), E2F1 (sc-193; Santa Cruz Biotechnology), and STAT-1 (G16920; Transduction Laboratories, BD Biosciences, Mississauga, CA), polyclonal Abs directed against a peptide from the C-terminal part of the PARP-1 automodification domain (422), or against PAR (LP-9610 or 10-H antibodies), or a mouse monoclonal antibody (C-2-10) raised against bovine PARP (both the 96-10 and C-2-10 Abs were bought from Dr. Guy Poirier, Unit of Health and Environment, CHUL Research Center, Québec, Canada). Membranes were also blotted with a mouse monoclonal antibody directed against β-actin (CLT9001, Jackson Immuno Research laboratories inc.; 1:35000 dillution), which has been used for normalization purposes. The blot was then washed and incubated with a 1:1000 dilution of a peroxidase-conjugated goat anti-rabbit (for the Sp1, Sp3, NFI, PARP-1 (422), p(ADP)r (LP-9610)) or anti-mouse (for the PARP-1 C-2-10 Ab) immunoglobulin G (Jackson Immunoresearch Lab.) and immunoreactive complexes revealed using a Western blot Detection Kit (Amersham, Baie d'Urfé, Canada). Each Western blot result shown in this study corresponds to one out of at least three representative experiments.
Nuclear extracts and electrophoretic mobility shift assay (EMSA)
Crude nuclear extracts were prepared as described  from both the PARP-1+/+ and PARP-1-/-cell lines and dialyzed against DNaseI buffer (50 mM KCl, 4 mM MgCl2, 20 mM K3PO4 (pH 7.4), 1 mM β-mercaptoethanol, 20% glycerol). Extracts were kept frozen in small aliquots at -80°C until use. EMSAs were carried out by incubating 3 × 104cpm of 5' end-labeled (32P-γATP) double-stranded oligonucleotides bearing the high affinity binding sites for the transcription factors Sp1, NFI, AP-1, E2F1, and STAT-1 with either 5- (for the Sp1 probe) or 10 μg (for the NFI, AP-1, E2F1 and STAT-1 probes) nuclear proteins in the presence of 25 ng poly(dI-dC). poly(dI-dC) (Pharmacia-LKD) in buffer D (5 mM HEPES; 10% glycerol; 0,05 mM EDTA; 0,125 mM PMSF). When indicated, unlabeled double-stranded oligonucleotides bearing various DNA target sequences for known transcription factors (Sp1, NFI) were added as unlabeled competitors (100- and 500-fold molar excesses) during the assay. The EMSA experiment with the Sp1 from the in vitro PARP assay (16 μl from the reaction mix was used for each lane) was conducted as above except that poly(dI-dC) was omitted and that DNA-protein complexes were separated on a 6% native polyacrylamide gel. Supershift experiments in EMSA were conducted by adding antibodies (400 ng) directed against Sp1, Sp3 and NFI to the above reaction mixtures. Incubation proceeded at room temperature for 5 min upon which time DNA-protein complexes were separated by gel electrophoresis through a 8% (for Sp1, AP-1, E2F1 and STAT-1) or 10% (for NFI) native polyacrylamide gel run against Tris-glycine buffer as described . Gels were dried and autoradiographed at -80°C to reveal the position of the shifted DNA-protein complexes.
The immunoprecipitation of the Sp1 complexes was performed as follow: approximately 300 μg nuclear extracts from the PARP-1+/+ and PARP-1-/- cell lines was mixed with either the anti-Sp1 antibody (2 μg; Santa Cruz) or normal rabbit IgG (2 μg; Santa Cruz) overnight at 4°C, either alone, or in the presence of 100 μg/ml ethidium bromide [22, 106], in binding buffer (10 mM Tris-HCl, 20 mM MgCl2, 150 mM NaCl, 1 mM dithiothreitol, 10% (v/v) glycerol, 1 mM PMSF). Protein-A-Sepharose (Sigma-Aldrich) was then added to the mixtures containing the cell extracts and incubated further for 5 h at 4°C. Samples were centrifuged and the protein A-Sepharose pellet washed four times in binding buffer before being resuspended in SDS sample buffer (6 M urea, 63 mM Tris (pH 6,8), 10% (vol/vol) glycerol, 1% SDS, 0,00125% (wt/vol) bromophenol blue, 300 mM β-mercaptoethanol). The immunoprecipitated samples were then used for Western blot analyses using the Sp1 (sc-59), PARP-1 (C-2-10) and PAR (LP-9610) antibodies. To immunoprecipitate the PARP-1 complexes, PARP-1+/+ and PARP-1-/- cells were cultured as described above and then harvested in lysis buffer (1% (v/v) NP-40, 175 mM KPO4, 150 mM NaCl, 1 mM dithiothreitol, 0,5 mM PMSF). The cell lysates were incubated for 1 h at 4°C and then cleared by centrifugation at 13,000 rpm for 10 min at 4°C. The anti-PARP-1 polyclonal antibody F-123 (100 μl) attached to protein A-Sepharose was then added to the cell lysates and the reaction mix incubated for 2 h at 4°C. The beads were pelleted by centrifugation, washed four times in lysis buffer and finally resuspended in SDS sample buffer. The immunoprecipitated samples were then used for Western blot analyses using the Sp1 (sc-59), Sp3 (sc-644), PARP-1 (422) and PAR (LP-9610) antibodies.
In vitro Poly(ADP-ribosyl)ation assay
Recombinant Sp1 protein (250 ng GST-Sp1-8xHis protein; kindly provided by Dr. Claude Labrie, Oncology and Molecular Endocrinology Research Center, CHUL) was incubated for 5 min at 30°C with 1 U of purified bovine PARP-1 in a standard assay mixture (152 μl) containing 100 mM Tris-HCl, pH 8.0, 10 mM MgCl2, 10% (v/v) glycerol, 1.5 mM DTT, 10 μg/ml activated DNA (DNaseI treated) and 200 μM NAD+ . The reaction was terminated by the addition of SDS sample buffer (6 M urea, 63 mM Tris (pH 6,8), 10% (vol/vol) glycerol, 1% SDS, 0,00125% (wt/vol) bromophenol blue, 300 mM β-mercaptoethanol), and the resulting mixture subjected to Western blot analysis with the PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610) antibodies. When indicated, the PARP inhibitor PJ34(Alexis Biochemicals, San Diego CA) was added to the reaction mixture at a final concentration of 100 mM along with purified PARP-1 15 min prior to the addition of the recombinant Sp1 protein.
Samples from the in vitro PARP assay were electrophoresed on 10% SDS-PAGE and electrotransfered onto nitrocellulose membranes at 50 V. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins transferred on the membranes was erased by incubation with poly(ADP-ribose) glycohydrolase (PARG) according to a previously described procedure . The blots were subsequently soaked in TS (150 mM NaCl, 10 mM Tris-HCl pH7.4) containing 0,1% (v/v) Tween-20 and 5% non-fat milk (TSM) for 1 hour and then soaked in a solution of 50 mM KPO4, pH 7,4, 50 mM KCl, 5 mM β-mercaptoethanol, 10% (v/v) glycerol, 0,1% (v/v) Triton X-100 containing 2,5 U/mL of purified calf thymus PARG  for 2 hours at room temperature. Membranes were washed four times with 10 mL of renaturation buffer (150 mM NaCl, 10 mM Tris-HCl pH7.4) at 15 min intervals. The membranes were finally blotted with antibodies directed against PARP-1 (C-2-10), Sp1 (sc-59) and PAR (LP-9610).
Transient transfection and CAT assay
PARP-1+/+ and PARP-1-/- cells were grown on tissue culture plates and transiently transfected 24 h later using the polycationic detergent Lipofectamine (Invitrogen-Gibco, On, Canada) following the procedure recommended by the manufacturer. When indicated, the PARP inhibitor PJ-34 (1 μM; Alexis Biochemicals) was added 3 h post-transfection. Each Lipofectamine-transfected plate received 1 μg of the PCR-CAT test plasmid and 1 μg pXGH5, which bears a secreted version of the human growth hormone gene upstream the mMT-I promoter . Levels of CAT activity for all transfected cells were determined as described  and normalized to the amount of hGH secreted into the culture media and assayed using a kit for quantitative measurement of hGH (Immunocorp, Montréal, Québec). The value presented for each individual test plasmid transfected corresponds to the mean of at least three separate transfections done in triplicate. Student's t-test was performed for comparison of the groups. To be considered significant, each individual value needed to be at least three times over the background level caused by the reaction buffer used (usually corresponding to 0.15% chloramphenicol conversion). Each single value was expressed as 100 × (% CAT in 4 h)/100 μg protein/ng hGH. Differences were considered to be statistically significant at P < 0.05. All data are expressed as mean ± SD.
The authors are grateful to Dr. Zhao-Qi Wang (Leibniz Institute for Age Research – Fritz Lipmann Institute, Jena, Germany) for his generous gift of both the A1 and F20 embryonic fibroblast cell lines, and Dr. Lucie Germain (Laboratoire d'Organogénèse Expérimentale (LOEX) Hôpital du St-Sacrement du CHA, Québec, Canada) for providing primary cultured, human skin keratinocytes. We also wish to thank both Dr. Girish Shaw (Laboratoire de recherche sur le cancer de la peau CHUQ, Centre de Recherche du Pavillon CHUL, Québec (Qc), Canada) and Dr. Masanao Miwa (Nagahama Institute of Bio-Science and Technology, Tsukuba-shi, Ibaraki, Japan) for their generous gift of the 10-H anti-polymer antibody. We also thank Steve Gagnon for his helpful technical assistance, and Jean-François Haince for providing the PARP-1 immunoprecipitation protocol (all from the Centre de Recherche du Pavillon CHUL, Québec (Qc), Canada). This work was supported by a grant from the National Science and Engineering Research Council of Canada (NSERC; grant OGP0138624) to S.L.G. K.Z. holds a Doctoral Research Award from the Vision network of the "Fonds de la Recherche en Santé du Québec" (FRSQ). S.D. and S.L.G. are research scholars (Junior and National levels, respectively) from the FRSQ.
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