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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

Model of interplay between PARP-1, Sp1 and other transcription factors. (A) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. (B) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. (C) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.
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Figure 9: Model of interplay between PARP-1, Sp1 and other transcription factors. (A) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. (B) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. (C) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.

Mentions: Although the reduced binding of Sp1 in wild-type PARP-1+/+ cells grown in the presence of the PARP activator H2O2 (Fig. 7A) is easily explained by the capacity of PARP-1 to add PAR to this transcription factor, the similar reduction observed in Sp1 expression (Fig. 1) and DNA binding (Fig. 2A) between both wild-type PARP-1+/+ and PARP-1-/- knockout cells remains speculative. However, the recent cloning of the human Sp1 gene promoter raises interesting possibilities to highlight the influence the lack of PARP-1 protein might have on the reduced expression of Sp1 (refer to Fig. 9 for additional details). Indeed, the basal promoter of the human Sp1 gene was found to bear target sites for multiple transcription factors, including NF-Y, AP-2, E2F, C/EBP as well as Sp1 itself [71]. Interestingly, the transcriptional activity directed by both AP-2 and E2F1 was found to be regulated by PARP-1 activity in cultured cells. PARP-1 apparently exerts a dual regulatory influence on AP-2α transcription with opposing effects: the middle region of the PARP-1 protein interacts physically with AP-2α and enhance its transcription, a situation postulated to occur under normal circumstances, whereas the catalytic domain strongly poly(ADP-ribosyl)ates AP-2α and thereby reduces binding to its DNA target sites, a temporary shut-off mechanism that might be used during unfavourable conditions [84]. On the other hand, E2F-1 was not found to be added PAR by PARP-1 but was found to physically interact with it, which results in an improved binding of E2F-1 to its DNA target sites and further enhances its trans-activating properties, and indicating that PARP-1 acts as a positive co-factor of E2F-1-mediated transcription [85]. Although binding of AP-2 to the Sp1 gene promoter could not be demonstrated, E2F was found to bind and positively influence transcription of that gene [71]. Therefore, the reduced expression of Sp1 that we observed in PARP-1-/- knockout cells may simply result from the reduced transcriptional activity of E2F-1 (and maybe also of AP-2) as the Sp1 gene has been reported to be positively regulated in a dose-dependent manner by this transcription factor [71] (Fig. 9).


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)

Model of interplay between PARP-1, Sp1 and other transcription factors. (A) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. (B) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. (C) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Model of interplay between PARP-1, Sp1 and other transcription factors. (A) PARP-1 plays a suppressive function (indicated by 'T' bars) on the DNA binding properties of Sp1, and indirectly, on its expression as well, by the enzymatic addition of poly(ADP-ribose) units (PAR) to Sp1. PARP-1 may exert its effect by stimulating the transcriptional properties (indicated by arrows) of both AP-2 and E2F-1 by physically interacting with these transcription factors (and therefore, independently of addition of PAR), of which the latter was recognized as a component required to ensure proper transcription of the human Sp1 gene. (B) Once PARP-1 is stimulated by DNA damages, post-translational modification of both Sp1 and AP-2 is increased to the point that their DNA binding properties and thereby, their transcriptional capacity, is considerably decreased without significantly altering their level of expression. (C) However, in the absence of PARP-1, addition of PAR is abrogated and the transcriptional capacity of Sp1 becomes dramatically increased despite that its overall expression is considerably reduced primarily as a consequence of: i) a reduction in both the expression [112] and the positive transcriptional influence of E2F1 [85], a property that requires a physical interaction with PARP-1, and ii) a reduced transcriptional activity of AP-2, which also requires a physical association of this transcription factor with the middle region of PARP-1 [84]. TrC: transcriptional capacity of Sp1; Exp: level of Sp1 expression.
Mentions: Although the reduced binding of Sp1 in wild-type PARP-1+/+ cells grown in the presence of the PARP activator H2O2 (Fig. 7A) is easily explained by the capacity of PARP-1 to add PAR to this transcription factor, the similar reduction observed in Sp1 expression (Fig. 1) and DNA binding (Fig. 2A) between both wild-type PARP-1+/+ and PARP-1-/- knockout cells remains speculative. However, the recent cloning of the human Sp1 gene promoter raises interesting possibilities to highlight the influence the lack of PARP-1 protein might have on the reduced expression of Sp1 (refer to Fig. 9 for additional details). Indeed, the basal promoter of the human Sp1 gene was found to bear target sites for multiple transcription factors, including NF-Y, AP-2, E2F, C/EBP as well as Sp1 itself [71]. Interestingly, the transcriptional activity directed by both AP-2 and E2F1 was found to be regulated by PARP-1 activity in cultured cells. PARP-1 apparently exerts a dual regulatory influence on AP-2α transcription with opposing effects: the middle region of the PARP-1 protein interacts physically with AP-2α and enhance its transcription, a situation postulated to occur under normal circumstances, whereas the catalytic domain strongly poly(ADP-ribosyl)ates AP-2α and thereby reduces binding to its DNA target sites, a temporary shut-off mechanism that might be used during unfavourable conditions [84]. On the other hand, E2F-1 was not found to be added PAR by PARP-1 but was found to physically interact with it, which results in an improved binding of E2F-1 to its DNA target sites and further enhances its trans-activating properties, and indicating that PARP-1 acts as a positive co-factor of E2F-1-mediated transcription [85]. Although binding of AP-2 to the Sp1 gene promoter could not be demonstrated, E2F was found to bind and positively influence transcription of that gene [71]. Therefore, the reduced expression of Sp1 that we observed in PARP-1-/- knockout cells may simply result from the reduced transcriptional activity of E2F-1 (and maybe also of AP-2) as the Sp1 gene has been reported to be positively regulated in a dose-dependent manner by this transcription factor [71] (Fig. 9).

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