Abstract
BackgroundPoly(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.ResultsExpression 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.ConclusionOur 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.
Highlights
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
Expression and DNA binding of Sp1, Sp3 and Nuclear Factor I (NFI) in PARP1+/+ and PARP-1-/- cells To investigate whether suppression of PARP-1 activity would result in alterations in the level of expression and/ or the DNA binding activity of the transcription factors that are critical for the transcription of the PARP-1 gene, crude nuclear extracts were prepared from embryonic fibroblast cell lines derived from both normal (PARP-1+/ +) and PARP-1 knockout mice (PARP-1-/-) [52]
Whereas no alteration was observed in the level of Sp3 expression between both types of cells, a dramatic reduction was observed for Sp1 in PARP-1-/- cells
Summary
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. Poly(ADP-ribose) polymerase-1 (PARP-1) is a highly evolutionary preserved, multifunctional nuclear enzyme that catalyzes the addition of ADP-ribose (ADPr) units from nicotinamide adenine dinucleotide (NAD+) on a large variety of nuclear proteins and impinges on many of the major nuclear functions (reviewed in [1]). Gene inactivation studies have revealed and/or confirmed up to nine biological functions for PARP-1 [2] These include both DNA repair and maintenance of genomic integrity as well as regulation of telomerase activity [3,4] ( reviewed in [5]). PARP-1 may contribute to the regulation of cytoskeletal organization and in the post-transcriptional modification of nuclear proteins like histones and transcription factors (reviewed in [2,12])
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