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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 K, Desnoyers S, Leclerc S, Guérin SL - BMC Mol. Biol. (2007)

Bottom Line: Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oncology and Molecular Endocrinology Research Center, Centre de Recherche du CHUL-CHUQ and Département d'Anatomie-Physiologie, Université Laval, Québec, G1V 4G2, Canada. Karine.zaniolo@crchul.ulaval.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

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PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro. (A) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was 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 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). (B) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD+/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.
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Figure 6: PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro. (A) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was 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 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). (B) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD+/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.

Mentions: As the PAR Ab revealed the presence of a poly(ADP-ribosyl)ated protein in the Sp1 immunoprecipitate that has the same electrophoretic mobility as that of Sp1, we therefore used in vitro PARP assays to determine whether PARP-1 could add PAR to this transcription factor. Western blot analyses were conducted to monitor both the PARP-1 and Sp1 proteins, as well as their poly(ADP-ribosyl)ation by the addition of PAR. PARP-1 alone possesses no intrinsic activity in the absence of its substrate NAD+ (Figure 6A, lane 1). However, the addition of 200 μM NAD+ was sufficient to turn on PARP-1 activity, which then added PAR to itself (Figure 6A; PARP-1Mod: bottom panel) through its automodification domain. Automodification of PARP-1, which dramatically changed its electrophoretic mobility in SDS-PAGE (yielding a smear on the gel; Figure 6A, lane 2: top panel), was entirely prevented when the PARP-1 inhibitor PJ34 was added to the reaction mix (Figure 6A, lane 3). As shown on lane 4, recombinant Sp1 is initially free of PAR. However, upon incubation with both PARP-1 and NAD+, a faint band with a molecular mass corresponding to Sp1 (Sp1Mod) could be detected with the PAR Ab just beneath the signal (smear) corresponding to the automodified PARP-1 (Figure 6A, lane 5: bottom panel). Again, the addition of the PJ34 PARP-1 inhibitor entirely prevented the addition of PAR to both proteins (PARP-1 and Sp1; lane 6). In order to demonstrate the specific addition of PAR to both Sp1 and PARP-1, both recombinant proteins were incubated together along with NAD+ as in lane 5. Protein samples were electrophoresed on SDS-PAGE and transferred on membranes as above. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins transferred on the membranes was then erased by incubation with PARG. As shown on Figure 6A (lane 8: bottom panel), no polymer could be detected by the PAR Ab on both the PARP-1 and Sp1 proteins upon exposure to PARG. The fact that the proteins were first fractionated on the SDS gel and membrane-transferred prior to the treatment with PARG explains the lack of any change in the electrophoretic mobility of PARP-1 on gel (lane 8, top panel), which again appears as a smear due to the initial addition of PAR.


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 K, Desnoyers S, Leclerc S, Guérin SL - BMC Mol. Biol. (2007)

PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro. (A) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was 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 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). (B) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD+/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.
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Figure 6: PARP-1-dependent poly(ADP-ribosyl)ation of Sp1 in vitro. (A) Recombinant Sp1 protein was incubated in reaction buffer either alone (lane 4) or with purified bovine PARP-1 (1 unit) in the presence of 200 μM NAD+ (lane 5). The reaction mixture was 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 was added to the reaction mixture with purified PARP-1 alone (lane 3) or in the presence of recombinant Sp1 (lane 6). When indicated, samples from the in vitro PARP assay were electrophoresed and electrotransfered onto nitrocellulose membranes. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins was then erased by incubation with PARG and the proteins analyzed by Western blotting with the same antibodies as detailed above (lane 8). Lane 1: PARP-1 alone; lane 2: PARP-1 incubated with NAD+; lane 3: same as in lane 2 plus PJ34; lane 7: same as in lane 5 but incubated in PARG buffer without addition of PARG-1. The position of modified PARP-1 (PARP-1Mod) and Sp1 (Sp1Mod) is indicated (left) along with the appropriate molecular mass marker (right). (B) Recombinant Sp1 was incubated in reaction buffer containing 200 μM NAD+ and nicked DNA either alone (+SP1; lane 3) or with purified bovine PARP-1 (1 unit) (+Sp1/PARP-1; lane 4). A sample (16 μl) from the reaction mixture was then incubated with the 5'-end labeled Sp1 oligonucleotide and formation of DNA-protein complexes monitored by EMSA as in Figure 2. As a control, the PARP-1 inhibitor PJ34 was added to the reaction mixture containing PARP-1/NAD+/Sp1 (+Sp1/PARP-1/PJ34; lane 5). Lane 1: labeled probe alone in reaction mix (P); Lane 2: labeled probe incubated in buffer D with PARP-1 but in the absence of NAD and Sp1 (+PARP-1). The position of both the Sp1 complex (Sp1) and the free probe (U) is indicated.
Mentions: As the PAR Ab revealed the presence of a poly(ADP-ribosyl)ated protein in the Sp1 immunoprecipitate that has the same electrophoretic mobility as that of Sp1, we therefore used in vitro PARP assays to determine whether PARP-1 could add PAR to this transcription factor. Western blot analyses were conducted to monitor both the PARP-1 and Sp1 proteins, as well as their poly(ADP-ribosyl)ation by the addition of PAR. PARP-1 alone possesses no intrinsic activity in the absence of its substrate NAD+ (Figure 6A, lane 1). However, the addition of 200 μM NAD+ was sufficient to turn on PARP-1 activity, which then added PAR to itself (Figure 6A; PARP-1Mod: bottom panel) through its automodification domain. Automodification of PARP-1, which dramatically changed its electrophoretic mobility in SDS-PAGE (yielding a smear on the gel; Figure 6A, lane 2: top panel), was entirely prevented when the PARP-1 inhibitor PJ34 was added to the reaction mix (Figure 6A, lane 3). As shown on lane 4, recombinant Sp1 is initially free of PAR. However, upon incubation with both PARP-1 and NAD+, a faint band with a molecular mass corresponding to Sp1 (Sp1Mod) could be detected with the PAR Ab just beneath the signal (smear) corresponding to the automodified PARP-1 (Figure 6A, lane 5: bottom panel). Again, the addition of the PJ34 PARP-1 inhibitor entirely prevented the addition of PAR to both proteins (PARP-1 and Sp1; lane 6). In order to demonstrate the specific addition of PAR to both Sp1 and PARP-1, both recombinant proteins were incubated together along with NAD+ as in lane 5. Protein samples were electrophoresed on SDS-PAGE and transferred on membranes as above. The PAR covalently linked onto the automodified PARP-1 and Sp1 proteins transferred on the membranes was then erased by incubation with PARG. As shown on Figure 6A (lane 8: bottom panel), no polymer could be detected by the PAR Ab on both the PARP-1 and Sp1 proteins upon exposure to PARG. The fact that the proteins were first fractionated on the SDS gel and membrane-transferred prior to the treatment with PARG explains the lack of any change in the electrophoretic mobility of PARP-1 on gel (lane 8, top panel), which again appears as a smear due to the initial addition of PAR.

Bottom Line: Expression of both Sp1 and NFI was found to be considerably reduced in PARP-1-/- cells.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Oncology and Molecular Endocrinology Research Center, Centre de Recherche du CHUL-CHUQ and Département d'Anatomie-Physiologie, Université Laval, Québec, G1V 4G2, Canada. Karine.zaniolo@crchul.ulaval.ca

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus