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

Karine Zaniolo 1 Serge Desnoyers 0 Steeve Leclerc 1 Sylvain L Gurin 1 0 Unite de Recherche en Pediatrie, Centre de Recherche du CHUL-CHUQ and Departement de Pediatrie, Universite Laval , Quebec, G1V 4G2 , Canada 1 Oncology and Molecular Endocrinology Research Center, Centre de Recherche du CHUL-CHUQ and Departement d'Anatomie- Physiologie, Universite Laval , Quebec, G1V 4G2 , Canada 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 PARP1 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 DNAindependent 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 PARP1 promoter as their mutation entirely abolished the increased promoter activity observed in PARP1-/- 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. - Background 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 consequently 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] (also reviewed in [5]). PARP-1 also regulates the expression of various proteins at the transcriptional level (reviewed in [2]) and is also involved in DNA replication as well as cell differentiation [6-8]. In addition, polymer of ADP ribose (PAR) has been recently identified as an emergency source of energy used by the base-excision machinery to synthesize ATP [9,10]. PAR may also serve as a signal to induce cell death [11]. Finally, 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]). PARP-1 plays vital functions in gene transcription as it can influence the state of chromatin remodeling through the catalytic addition of PAR to the core histones [13,14]. Besides, PARP-1, through its double Zn-finger DNA binding domain, can interact with target regulatory elements present in the promoter of many genes, including the MCAT 1 [15], Pax-6 [16], MHC II [17], and the CXCL1 [18] genes. By exploiting both chromatin cross-linking and immunoprecipitation assays, Soldatenkov and coworkers elegantly demonstrated that PARP-1 could also bind to secondary hairpin-like structures present in the 5'flanking region of the human PARP-1 promoter [19]. Although no prototypical sequence that could be recognized as a target by the PARP-1 DBD has been identified yet, Pion and collaborators have however demonstrated the ability of the protein to bind to both 5'- and 3'recessed ends on double-stranded DNA, as well as to palindromic-like structures often present in DNA [20]. Lastly, PARP-1 can also alter gene transcription through its ability to poly(ADP-ribosyl)ate several transcription factors, such as YY1 [21], NFKB [22], TFIIF [23], Oct-1 [24], B-MYB [25] and AP-2 [26], thereby preventing their binding with their specific promoter target sites [27]. The studies conducted over the last few decades have been mostly dedicated to the study of the many biological and cellular functions of PARP-1. Unfortunately, not so many studies have explored the molecular mechanisms that regulate the transcription of PARP-1 gene expression. Unlike its enzymatic activity, PARP-1 gene transcription is not activated by DNA strand breaks [28,29]. On the other hand, PARP-1 gene expression appears related to cell proliferation rather than DNA synthesis as PARP-1 mRNA has been shown to be more abundant in the G1 phase of the cell cycle [30-32]. To date, the PARP-1 promoter has been cloned from three different mammalian species: human [33], rat [34] and mouse [35]. All three mammalian basal promoters share structural similarities typical of housekeeping genes in that they lack a functional consensus TATA box, possess a high content of GC-residues, and bear a consensus initiator sequence (Inr) that overlaps the transcription initiation site. The human promoter has been shown to contain binding sites for the transcription factors Sp1, AP-2 [33], YY1 [21], and Ets [36], whereas the mouse promoter was recently shown to be down-regulated by a complex of adenovirus E1A protein and pRb [35]. Studies that we conducted on the rat PARP-1 (rPARP-1) proximal promoter have shown that its activity is primarily, but not entirely dependent on the recognition of five GC-rich binding sites (F1, F2, F3, F4 and US1) by the positive transcription factors Sp1 and Sp3 [37,38]. Other transcription factors, such as those that belong to the Nuclear Factor I (NFI) family of transcription factors [39], were also found to bind to nearby target sites to alter the activity directed by the PARP-1 gene promoter [39-41]. Sp1 and Sp3, two members from a Zn-finger family of transcription fac (...truncated)