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Expression of the gene encoding poly(ADP-ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US-1 and F1ϪF4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site-directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions Ϫ101 and Ϫ60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T-lymphoblastoid and Ltk Ϫ fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site-directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis-acting elements by transcription factors other than Sp1.Keywords : poly(ADP-ribose) polymerase; Sp1 ; electrophoretic mobility-shift assay; transcription factor; gene control.Poly(ADP-ribose) polymerase (PARP) is a 116-kDa eukary-organ to be exposed to DNA-damaging agents. PARP is also thought to participate in the biochemical changes that accomotic nuclear enzyme that catalyses the transfer of ADP-ribose pany apoptosis [6]. During programmed cellular death, the units from NAD ϩ to a limited number of nuclear acceptor pro-PARP enzyme is cleaved by the protease resembling ICE teins involved in chromatin architecture and DNA metabolism, (PRICE) [7], this proteolytic cleavage being considered an early including PARP itself [1, 2]. Its activation is triggered by DNAmarker of the apoptotic state [6]. strand breaks introduced in the genome through environmentalThe human PARP (hPARP) cDNA has been isolated [8Ϫ injuries. This enzyme is made up of three functional domains : 11] and the corresponding gene located on the q42 region of an amino-terminal fragment containing the DNA-binding region chromosome 1 [12]. It constitutes 23 exons extending over that comprises two zinc fingers required for the recognition of 43 kbp of DNA sequence [8]. Its promoter contains potential DNA-strand breaks, a central domain including the automodifiSp1 binding sites and two assortments of inverted repeats. cation sites, and a carboxy-terminal fragment bearing the cata-CCAAT and TATA boxes [13,14] were also identifi...

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Poly(ADP-ribose) polymerase-1 (PARP-1) catalyzes the rapid and extensive poly(ADP-ribosyl)ation of nuclear proteins in response to DNA strand breaks, and its expression, although ubiquitous, is modulated from tissue to tissue and during cellular differentiation. PARP-1 gene promoters from human, rat, and mouse have been cloned, and they share a structure common to housekeeping genes, as they lack a functional TATA box and contain multiple GC boxes, which bind the transcriptional activator Sp1. We have previously shown that, although Sp1 is important for rat PARP1 (rPARP) promoter activity, its finely tuned modulation is likely dependent on other transcription factors that bind the rPARP proximal promoter in vitro. In this study, we identified one such factor as NF1-L, a rat liver isoform of the nuclear factor 1 family of transcription factors. The NF1-L site on the rPARP promoter overlaps one of the Sp1 binding sites previously identified, and we demonstrated that binding of both factors to this composite element is mutually exclusive. Furthermore, we provide evidence that NF1-L has no effect by itself on rPARP promoter activity, but rather down-regulates the Sp1 activity by interfering with its ability to bind the rPARP promoter in order to modulate transcription of the rPARP gene.Poly(ADP-ribose) polymerase-1 (PARP-1) 1 is a nuclear enzyme which catalyzes the addition of ADP-ribose units from nicotinamide adenine dinucleotide (NAD ϩ ) onto itself and other nuclear proteins such as histones and topoisomerases (reviewed in Refs. 1 and 2). It is made up of three functional domains, namely the amino-terminal DNA-binding domain, the central automodification domain, and the carboxyl-terminal catalytic domain (3). Although PARP-1 is often associated with DNA repair, because of its rapid and extensive activation following DNA damage (4, 5), it has also been implicated in other major nuclear functions such as transcription (6, 7), DNA replication (8, 9) and recombination (10). PARP-1 is also important for cell differentiation (11-13) and is involved in cell death, likely acting as a molecular switch between apoptosis and necrosis (14).PARP-1 is expressed in all organs, albeit at varying degrees, with highest mRNA expression found in brain, thymus, heart, and testis (15, 16). PARP-1 mRNA level is regulated at the cell cycle level, reaching its peak at either the G 1 (17-20) or the S phases (21, 22). It has also been shown that a decrease in PARP-1 mRNA levels is associated with cellular differentiation (23-26) and senescence (27), whereas an increase is observed upon activation of lymphocytes (17, 28) or peripheral blood mononuclear cells (18). All these studies show that, although PARP-1 is ubiquitously expressed, its modulation, likely through complex transcriptional regulation, is critical to major cellular functions.In order to better understand the transcriptional mechanisms regulating PARP-1 expression, the PARP-1 gene promoter has been identified and cloned from three mammalian species, human (29, 30), rat (31), ...

Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors.