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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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DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1+/+ and PARP-1-/- cells. (A) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. (B) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. (C) Nuclear extracts (10 μg) from both PARP-1+/+ and PARP-1-/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).
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Figure 3: DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1+/+ and PARP-1-/- cells. (A) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. (B) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. (C) Nuclear extracts (10 μg) from both PARP-1+/+ and PARP-1-/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).

Mentions: To eliminate the possibility that a widespread suppression of all transcription factors might have accounted for the drop in both Sp1 and NFI expression in PARP-1-/- knockout cells, we investigated the expression and DNA binding properties of transcription factors unrelated to the transcription of the PARP-1 gene. Double-stranded oligonucleotides bearing the target sites for the transcription factors AP-1, E2F1 and STAT-1 were therefore 5'-end labeled and used in EMSA in combination with nuclear extracts obtained from both PARP-1+/+ and PARP-1-/- cells. As shown on Figure 3, whereas binding of AP-1 to its prototypical target site was entirely abolished in PARP-1-/- cells, that of both E2F1 and STAT-1 remained unaffected by the lack of PARP-1 expression in the knockout cells. Western blot analyses indicated that the lack of AP-1 binding resulted from the absence of c-jun in the PARP-1-/- cells, whereas no change was observed in the expression of both the E2F1 and STAT-1 proteins. Therefore, the reduction in the expression and DNA binding of both Sp1 and NFI is closely linked to the lack of functional PARP-1 in the PARP-1-/- knockout cells and is not merely the consequence of a general shutdown of gene expression that might have occurred in these cells.


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)

DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1+/+ and PARP-1-/- cells. (A) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. (B) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. (C) Nuclear extracts (10 μg) from both PARP-1+/+ and PARP-1-/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175517&req=5

Figure 3: DNA binding and expression of the transcription factors AP-1, E2F1 and STAT-1 in PARP-1+/+ and PARP-1-/- cells. (A) EMSA analysis. Crude nuclear proteins (5 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with a 5' end-labeled probe bearing the high affinity binding site for AP-1, E2F1 and STAT-1. Formation of DNA/protein complexes was then monitored by EMSA on an 8% gel as detailed in Figure 2. The position of the AP-1, E2F1 and STAT-1 DNA-protein complexes is shown, as well as that of the free probe (U). P: labeled probe alone. (B) Coomassie blue staining of the protein samples used for conducting both the EMSA (panel A) and the Western blot experiment (panel C). One protein band present in both extract was randomly selected and its intensity determined by densitometric analysis in order to precisely calibrate the protein concentration used for the assays. (C) Nuclear extracts (10 μg) from both PARP-1+/+ and PARP-1-/- cells were examined in Western blot as in Figure 1 using antibodies directed against E2F1, STAT-1 and the AP-1 subunit c-jun. The position of the nearest molecular mass markers is indicated (60 kDa and 85 kDa).
Mentions: To eliminate the possibility that a widespread suppression of all transcription factors might have accounted for the drop in both Sp1 and NFI expression in PARP-1-/- knockout cells, we investigated the expression and DNA binding properties of transcription factors unrelated to the transcription of the PARP-1 gene. Double-stranded oligonucleotides bearing the target sites for the transcription factors AP-1, E2F1 and STAT-1 were therefore 5'-end labeled and used in EMSA in combination with nuclear extracts obtained from both PARP-1+/+ and PARP-1-/- cells. As shown on Figure 3, whereas binding of AP-1 to its prototypical target site was entirely abolished in PARP-1-/- cells, that of both E2F1 and STAT-1 remained unaffected by the lack of PARP-1 expression in the knockout cells. Western blot analyses indicated that the lack of AP-1 binding resulted from the absence of c-jun in the PARP-1-/- cells, whereas no change was observed in the expression of both the E2F1 and STAT-1 proteins. Therefore, the reduction in the expression and DNA binding of both Sp1 and NFI is closely linked to the lack of functional PARP-1 in the PARP-1-/- knockout cells and is not merely the consequence of a general shutdown of gene expression that might have occurred in these cells.

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