Limits...
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.

Show MeSH

Related in: MedlinePlus

Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1+/+ and PARP-1-/- cells. (A) Immunoprecipitation of the Sp1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. (B) Immunoprecipitation of the PARP-1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175517&req=5

Figure 4: Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1+/+ and PARP-1-/- cells. (A) Immunoprecipitation of the Sp1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. (B) Immunoprecipitation of the PARP-1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.

Mentions: As PARP-1 has been shown to physically interact with many nuclear proteins of which some are transcription factors, we then wished to determine whether any of Sp1, Sp3 or NFI could represent a target for PARP-1 in vitro. Sp1 was therefore immunoprecipitated from nuclear extracts prepared from PARP+/+ and PARP-/- cells using the Sp1 Ab (sc-59) and the immunoprecipitated proteins analyzed on Western blot with Abs directed against either Sp1 or PARP-1 (C-2-10). As shown on Figure 4A, Sp1 was very efficiently immunoprecipitated with the Sp1 Ab as it could be detected in the extracts from both the PARP-1+/+ and PARP-1-/- cells. As expected, a much weaker Sp1 signal was obtained with the PARP-1-/- nuclear extract. Western blotting the Sp1-immunoprecipitated proteins with the PARP-1 Ab revealed clearly the presence of PARP-1 in the extract from PARP-1+/+ cells but not in that from PARP-1-/- cells, suggesting that indeed, Sp1 and PARP-1 can physically interact with each other. As expected, no signal was observed when either protein A-Sepharose (Ctl-) or a rabbit IgG Ab were added as negative controls to the extract in the absence of Sp1 Ab. Blotting of the membrane with the LP-9610 Ab against PAR revealed the presence of two poly(ADP-ribosyl)lated proteins in the extract from PARP-1+/+ but not PARP-1-/- cells: a more intense band with an electrophoretic mobility identical to that corresponding to PARP-1, and a weaker, faster-migrating band with a mobility on gel identical to that expected for Sp1. Again, both negative controls (protein A-Sepharose (Ctl-) and rabbit IgG Ab) yielded no signal at a position similar to those seen with the Sp1 Ab. As a positive control, total proteins were prepared from E. coli cells transformed with a recombinant plasmid that encodes high levels of a fully functional, truncated PARP-1 [55], and used in Western blotting. As shown on Figure 4A, blotting of the bacterially produced recombinant PARP-1 protein with the PAR Ab LP-9610 revealed a smear that is typical of PAR-modified proteins [56].


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)

Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1+/+ and PARP-1-/- cells. (A) Immunoprecipitation of the Sp1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. (B) Immunoprecipitation of the PARP-1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Co-immunoprecipitation of Sp1 and PARP-1 in protein extracts from PARP-1+/+ and PARP-1-/- cells. (A) Immunoprecipitation of the Sp1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Crude nuclear proteins (300 μg) from both PARP-1+/+ and PARP-1-/- cells were incubated with the Sp1 Ab (sc-59) and the Sp1-protein complexes recovered by the addition of protein-A-Sepharose. The resulting immunoprecipitated proteins were then SDS-gel fractionated before being membrane-transferred and Western blotted with antibodies against Sp1, PARP-1 (C-2-10) and PAR (LP-9610). Ctl-: protein A-Sepharose added to crude nuclear proteins in the absence of Sp1 Ab and used as a negative control. IgG-Ab: normal rabbit IgG incubated with nuclear proteins prior to addition of protein A-Sepharose as a negative control. (B) Immunoprecipitation of the PARP-1-protein complexes in PARP-1+/+ and PARP-1-/- nuclear extracts. Same as in panel A except that the immunoprecipitation was conducted using the PARP-1 F-123 Ab. The blotted, PARP-1-immunoprecipitated proteins were then analyzed with the PARP-1 (422), Sp1 (sc-59), Sp3 (sc-644), and PAR (LP-9610) antibodies. Negative controls (Ctl- and IgG-Ab) are as in panel A. TE: total cell extract that has not been immunoprecipitated with the PARP-1 Ab.
Mentions: As PARP-1 has been shown to physically interact with many nuclear proteins of which some are transcription factors, we then wished to determine whether any of Sp1, Sp3 or NFI could represent a target for PARP-1 in vitro. Sp1 was therefore immunoprecipitated from nuclear extracts prepared from PARP+/+ and PARP-/- cells using the Sp1 Ab (sc-59) and the immunoprecipitated proteins analyzed on Western blot with Abs directed against either Sp1 or PARP-1 (C-2-10). As shown on Figure 4A, Sp1 was very efficiently immunoprecipitated with the Sp1 Ab as it could be detected in the extracts from both the PARP-1+/+ and PARP-1-/- cells. As expected, a much weaker Sp1 signal was obtained with the PARP-1-/- nuclear extract. Western blotting the Sp1-immunoprecipitated proteins with the PARP-1 Ab revealed clearly the presence of PARP-1 in the extract from PARP-1+/+ cells but not in that from PARP-1-/- cells, suggesting that indeed, Sp1 and PARP-1 can physically interact with each other. As expected, no signal was observed when either protein A-Sepharose (Ctl-) or a rabbit IgG Ab were added as negative controls to the extract in the absence of Sp1 Ab. Blotting of the membrane with the LP-9610 Ab against PAR revealed the presence of two poly(ADP-ribosyl)lated proteins in the extract from PARP-1+/+ but not PARP-1-/- cells: a more intense band with an electrophoretic mobility identical to that corresponding to PARP-1, and a weaker, faster-migrating band with a mobility on gel identical to that expected for Sp1. Again, both negative controls (protein A-Sepharose (Ctl-) and rabbit IgG Ab) yielded no signal at a position similar to those seen with the Sp1 Ab. As a positive control, total proteins were prepared from E. coli cells transformed with a recombinant plasmid that encodes high levels of a fully functional, truncated PARP-1 [55], and used in Western blotting. As shown on Figure 4A, blotting of the bacterially produced recombinant PARP-1 protein with the PAR Ab LP-9610 revealed a smear that is typical of PAR-modified proteins [56].

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