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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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Related in: MedlinePlus

rPARP-1 promoter activity in PARP-1+/+ and PARP-1-/- cells. (A) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1+/+ and PARP-1-/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test). S.D. is also provided. (B) The plasmids PCR3, -92α5CAT and α6–84 were transfected into both PARP-1+/+ and PARP-1-/- cells and CAT activity (expressed as the ratio of CAT activity from PARP-1-/- cells over that measured in PARP-1+/+ cells (considered as 100%)) measured and normalized as detailed above. Asterisks (*) correspond to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test).
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Figure 8: rPARP-1 promoter activity in PARP-1+/+ and PARP-1-/- cells. (A) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1+/+ and PARP-1-/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test). S.D. is also provided. (B) The plasmids PCR3, -92α5CAT and α6–84 were transfected into both PARP-1+/+ and PARP-1-/- cells and CAT activity (expressed as the ratio of CAT activity from PARP-1-/- cells over that measured in PARP-1+/+ cells (considered as 100%)) measured and normalized as detailed above. Asterisks (*) correspond to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test).

Mentions: Most of the activity directed by the PARP-1 gene promoter was shown to rely essentially on the recognition of multiple PARP-1 promoter target sites by members of the Sp1 family [37,38,41]. Of them, Sp1 accounted for most of the positive influence exerted on this promoter [54]. We demonstrated above that both expression and DNA binding of Sp1 is considerably decreased in PARP-1-/- cells. We therefore examined whether this decrease would also translate into a reduced PARP-1 promoter activity upon transfection of PARP-1-/- cells with a recombinant construct bearing the CAT reporter gene fused to the basal promoter from the rat PARP-1 gene that has its three Sp1 target sites (F2, F3 and F4) either kept intact (in pCR3) or mutated (in pCR3F2/F3/F4m) [37]. Unexpectedly, transfection of the pCR3 construct into PARP-1-/- cells yielded CAT activities approximately 4-fold higher than in PARP-1+/+ cells (Figure 8A). Consistent with these results, exposing pCR3-transfected PARP-1+/+cells that express the wild type PARP-1 protein to the PARP-1 inhibitor PJ34 resulted in a near 8-fold increase in basal PARP promoter activity while it had no influence in PARP-1-/- cells. Mutating all three Sp1 sites from the PARP-1 promoter (in pCR3F2/F3/F4m) dramatically reduced basal promoter activity and entirely suppressed induction of PARP-1 promoter function in PARP-1-/- cells. Suppression of PARP-1 activity with PJ34 in PARP-1+/+ cells had no influence on the activity directed by pCR3F2/F3/F4m suggesting that the regulatory influences of PARP-1 on its own promoter are mediated through alteration of Sp1.


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)

rPARP-1 promoter activity in PARP-1+/+ and PARP-1-/- cells. (A) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1+/+ and PARP-1-/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test). S.D. is also provided. (B) The plasmids PCR3, -92α5CAT and α6–84 were transfected into both PARP-1+/+ and PARP-1-/- cells and CAT activity (expressed as the ratio of CAT activity from PARP-1-/- cells over that measured in PARP-1+/+ cells (considered as 100%)) measured and normalized as detailed above. Asterisks (*) correspond to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test).
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Figure 8: rPARP-1 promoter activity in PARP-1+/+ and PARP-1-/- cells. (A) The recombinant plasmids PCR3 and PCR3F2/F3/F4m were transfected into both PARP-1+/+ and PARP-1-/- cells grown with or without the PARP-1 inhibitor PJ34. CAT activities were measured and normalized to the amount of hGH secreted into the culture medium. Values are expressed as ((%CAT activity/100 μg proteins)/ng hGH). Asterisks (*) indicate CAT activities from cells exposed to PJ34 that are statistically different from those measured when cells are transfected with pCR3 in the absence of inhibitor whereas † corresponds to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test). S.D. is also provided. (B) The plasmids PCR3, -92α5CAT and α6–84 were transfected into both PARP-1+/+ and PARP-1-/- cells and CAT activity (expressed as the ratio of CAT activity from PARP-1-/- cells over that measured in PARP-1+/+ cells (considered as 100%)) measured and normalized as detailed above. Asterisks (*) correspond to CAT activities in PARP-1-/- cells that are statistically different from those measured in PARP-1+/+ cells (P < 0.005; paired samples, t-test).
Mentions: Most of the activity directed by the PARP-1 gene promoter was shown to rely essentially on the recognition of multiple PARP-1 promoter target sites by members of the Sp1 family [37,38,41]. Of them, Sp1 accounted for most of the positive influence exerted on this promoter [54]. We demonstrated above that both expression and DNA binding of Sp1 is considerably decreased in PARP-1-/- cells. We therefore examined whether this decrease would also translate into a reduced PARP-1 promoter activity upon transfection of PARP-1-/- cells with a recombinant construct bearing the CAT reporter gene fused to the basal promoter from the rat PARP-1 gene that has its three Sp1 target sites (F2, F3 and F4) either kept intact (in pCR3) or mutated (in pCR3F2/F3/F4m) [37]. Unexpectedly, transfection of the pCR3 construct into PARP-1-/- cells yielded CAT activities approximately 4-fold higher than in PARP-1+/+ cells (Figure 8A). Consistent with these results, exposing pCR3-transfected PARP-1+/+cells that express the wild type PARP-1 protein to the PARP-1 inhibitor PJ34 resulted in a near 8-fold increase in basal PARP promoter activity while it had no influence in PARP-1-/- cells. Mutating all three Sp1 sites from the PARP-1 promoter (in pCR3F2/F3/F4m) dramatically reduced basal promoter activity and entirely suppressed induction of PARP-1 promoter function in PARP-1-/- cells. Suppression of PARP-1 activity with PJ34 in PARP-1+/+ cells had no influence on the activity directed by pCR3F2/F3/F4m suggesting that the regulatory influences of PARP-1 on its own promoter are mediated through alteration of Sp1.

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