Cellular Functions of PARP-1 - Human Poly(ADP-Ribose) Polymerase-1-Expressing Embryonic Stem Ce

Three non-exclusive mechanisms of the cellular functions of PARP-1 can be distinguished:

(i) Functions that rely on the enzymatic activity of PARP-1 and the subsequent covalent or non-covalent interaction of nuclear proteins with poly(ADP-ribose). (ii) Direct interactions of proteins with PARP-1 via protein-protein interaction, e.g. via the BRCT domain.

(iii) Intervention in the cellular NAD⁺ metabolism by excessive PARP-1 stimulation and potential signaling functions of free poly(ADP-ribose) or its derivatives. The consequences of these actions with regard to modulation of chromatin structure, genomic maintenance, transcription, and cell death are discussed in the following sections.

1.1.2.5.1 PARP-1 and Chromatin Modification

Chromatin is a complex of DNA and proteins with a dynamic structure involved in replication, transcription and other fundamental cellular processes (Felsenfeld and Groudine 2003). PARP-1 seems to act as a structural and regulatory component of chromatin, both in undamaged cells and upon genotoxic stress (Kim et al. 2004). Many poly(ADP-ribose) acceptor proteins were shown to contribute to chromatin and nuclear architecture such as histones, lamins, high-mobility group (HMG) proteins, topoisomerases, and the DEK protein (Figure 1.5) (Gagne et al. 2003; Rouleau et al. 2004; Ditsworth et al. 2007; Gamble and Fisher 2007).

Althaus and colleagues proposed a histone shuttle mechanism, based on the findings that poly(ADP-ribosyl)ation of polynucleosomes causes relaxation of chromatin structure and that activity of PARG degrades poly(ADP-ribose) from modified histones (Poirier et al. 1982; de Murcia et al.; Althaus 1992; Realini and Althaus 1992). According to this model, DNA-bound histones dissociate from DNA upon poly(ADP-ribosyl)ation, causing an open chromatin structure and guiding repair factors to sites of DNA damage. Upon degradation of the ADP-ribose polymer by PARG, DNA reassociates with histones, thereby restoring the condensed chromatin structure. Kim et al. reported that PARP-1 itself is a component of chromatin (Kim et al. 2004). They demonstrated in vitro that histone H1 and PARP-1 bind in a competitive and mutually exclusive manner to nucleosomes. Thereby, PARP-1 promotes the local compaction of chromatin into higher-order structures, which are associated with transcriptional repression.

Those authors suggested that PARP-1 modulates the chromatin architecture and gene transcription through its intrinsic enzymatic activity in a DNA-damage-independent manner; i.e.

PARP-1 automodification through a DNA-damage-independent trigger leads to its release from chromatin, thereby facilitating chromatin decondensation and gene transcription by RNA polymerase II. On the other hand, the same group recently demonstrated a reciprocal binding pattern of PARP-1 and histones H1 at many RNA polymerase II-transcribed promoters. Here, PARP-1 could replace histone H1 in a subset of these promoters, which was associated with actively transcribed genes (Krishnakumar et al. 2008). These findings suggest a functional interplay of PARP-1 with other chromatin-associated factors, implying an active role of PARP-1 in chromatin remodeling and transcriptional regulation. The detailed spatial and temporal characteristics of these mechanisms, however, remain to be determined.

Another mechanism of PARP-1-dependent chromatin regulation arises from the finding that poly(ADP-ribose) and automodified PARP-1 non-covalently interact with the 20S proteasome in the nucleus, which enhances its peptidase activity (Mayer-Kuckuk et al. 1999). It was proposed that this leads to the degradation of oxidatively damaged histones underscoring the function of PARP-1 in maintenance of nuclear stability (Ullrich et al. 1999).

1.1.2.5.2 PARP-1 and Genomic Maintenance

It was estimated that 20000 to 40000 DNA strand breaks occur in a mammalian cell per day, all of which need to be repaired to ensure genomic stability (Vijg 2007). In mammals, at least six, partly overlapping DNA repair pathways exist, i.e. O⁶-methyl guanine methyltransferase (MGMT), base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), and DNA double strand break (DSB) repair including the subpathways homologous recombination (HR) and non-homologous end joining (NHEJ) (Hoeijmakers 2001).

Figure 1.5: PARP-1, its interaction partners, and their role in genomic maintenance.

ATM indicates ataxia telangiectasia mutated; Bub3, Budding uninhibited by benzimidazoles 2; Cenpa/b, centromeric protein a/b; CSB, Cockayne syndrome type B; DEK, DEK oncogene; DNA-Polβ, DNA polymerase β;

DNA-PK_CS, DNA-activated protein kinase catalytic subunit; HMGB1, high mobility group box 1; Ku70/80, Ku antigens 70/80 kD subunit; MRE11, meiotic recombination 11; p21, cyclin-dependent kinase inhibitor 1A; p53, tumor protein p53; PCNA, proliferating cell nuclear antigen; TRF2, telomeric repeat binding factor 2; WRN, Werner syndrome protein; XRCC1, X-ray repair complementing defective in Chinese hamster 1; XPA, xeroderma pigmentosum complementation group A.

Except for the MGMT and MMR pathway, PARP-1 is involved in all of these repair pathways and is therefore considered as a general caretaker of genomic stability (Figure 1.5) (Meyer-Ficca et al. 2005a). It was shown that the recruitment of PARP-1 to sites of DNA damage, its activation, and the subsequent production of poly(ADP-ribose) is one of the first responses in mammalian DNA repair (D'Amours et al. 1999). Several studies supported the role of 1 as a cell survival factor upon genotoxic stimuli: Trans-dominant inhibition of PARP-1 by overexpression of its DNA binding domain potentiates cytotoxicity upon treatment of cells

with alkylating agents and ionizing radiation (Küpper et al. 1995). [N.B. ionizing radiation, such as X-irradiation or γ-irradiation, can introduce various forms of DNA damage, including oxidation of bases, DNA strand breaks and other lesions caused by oxygen radicals (Masutani et al. 2000)]. Moreover, PARP-1-deficient cells exhibit an enhanced sensitivity to alkylating agents (Trucco et al. 1998; Masutani et al. 1999a) and show increased frequencies of sister-chromatid exchanges, spontaneously or upon treatment with alkylating agents (de Murcia et al.

1997; Wang et al. 1997). Consistent with these results, Meyer et al. demonstrated in overexpression studies that PARP-1 acts as a negative regulator of alkylation-induced sister chromatid exchange (Meyer et al. 2000). However, in a different study, overexpression of hPARP-1 sensitized hamster cells to γ-irradiation, indicating an ambiguous role of PARP-1 in DNA damage response (Van Gool et al. 1997).

1.1.2.5.2.1 PARP-1 and its Interaction Partner PARP-2 in Base Excision Repair

Base excision repair is the major repair pathway, acting on damages that occur during cellular metabolism including damages from reactive oxygen species, methylation, deamination, and hydroxylation. The core BER reaction is initiated by a DNA single strand break (SSB) introduced by exogenous or endogenous factors (Hoeijmakers 2001). PARP-1 detects these SSB via its second zinc finger ZFII (Gradwohl et al. 1990; Molinete et al. 1993). Moreover, PARP-1 interacts with the BER loading platform X-ray repair complementing factor 1 (XRCC1) via its BRCT domain (Figure 1.5) (Masson et al. 1998; Dantzer et al. 1999; El-Khamisy et al. 2003). It was shown that PARP-1 is required for the assembly and stability of XRCC1 nuclear foci after DNA damage (El-Khamisy et al. 2003). Here, foci formation was also mediated via the interaction of poly(ADP-ribose) with XRCC1. Furthermore, XRCC1 and PARP-1 interact with DNA polymerase-β and DNA ligase III, forming a multiprotein complex consisting of the major BER factors (Caldecott et al. 1996; Leppard et al. 2003; Confer et al. 2004).

The finding that PARP-1 knock-out cells still synthesized poly(ADP-ribose) led to the identification of an additional nuclear PARP, PARP-2, which was also demonstrated to be activated upon genotoxic stimuli (Shieh et al. 1998; Ame et al. 1999; Bürkle 2006). Up to now, 1 and 2 are the only known DNA damage-dependent PARPs. 1 and PARP-2 homo- and heterodimerize (Figure 1.5) and work at least partly in a redundant fashion, since only double knock-out mice show embryonic lethality (section 1.1.2.6.1) (Schreiber et al. 2002;

Menissier de Murcia et al. 2003). This notion is supported by the fact that PARP-2 also participates in BER physically and functionally interacting with XRCC1, DNA polymerase-β, and DNA ligase III. Recent experiments using live cell imaging indicated a role of PARP-2 in later steps of BER repair, as proposed by the following model for spatio-temporal accumulation of BER factors: SSBs are detected by the DNA binding domain of PARP-1, leading to its activation, production of poly(ADP-ribose), and chromatin relaxation. Subsequently, additional

PARP-1 molecules are attracted, causing amplification of the signal. At the „point of repulsion‟

PARP-1 then dissociates from the DNA, enabling the recruitment of the BER loading platform XRCC1, PARP-2, and further DNA repair factors. This triggers resealing of the DNA lesion and re-establishment of genomic integrity (Mortusewicz et al. 2007).

1.1.2.5.2.2 PARP-1 in Nucleotide Excision Repair

Nucleotide excision repair is responsible for the removal of bulky DNA adducts, such as pyrimidine dimers, which are caused by UV irradiation (Hoeijmakers 2001).

Although the role of PARP-1 in NER is not very well established, at least two NER factors, the DNA-dependent ATPase Cockayne syndrome group B (CSB) protein and the DNA lesion recognition protein xeroderma pigmentosum group A (XPA), were identified as poly(ADP-ribose) binding enzymes (Figure 1.5) (Thorslund et al. 2005; Fahrer et al. 2007). CSB also physically interacts with PARP-1 and its ATPase activity is inhibited by poly(ADP-ribosyl)ation. Consistently, a third study suggested that PARP-1 is involved in repair of pyrimidine dimers in a CSB-dependent pathway (Flohr et al. 2003).

1.1.2.5.2.3 PARP-1 in Double Strand Break Signaling

DNA double strand breaks (DSBs) arise from ionizing radiation, free radicals, chemicals, or during replication of a SSB through collapsed replication forks. Mammalian cells repair DSBs via two mechanisms: homologous recombination (HR) utilizes the sister chromatid or chromosome for error-free repair of the lesion (for a detailed description of HR see section 1.2.1, Figure 1.9), whereas non-homologous enjoining (NHEJ) simply reattaches free DNA ends without using a template and is therefore error prone (Hoeijmakers 2001). The implementation of one of these pathways depends on the species, cell type, and cell cycle phase (Shrivastav et al. 2008).

In both pathways, PARP-1 already participates in very early phases. PARP-1 and the DSB sensing complexes MRE11/Rad50/NBS1 (involved in HR) and Ku70/80 (involved in NHEJ) were shown to interact with and compete for binding at free DNA ends, with PARP-1 potentially guiding these proteins to the damaged site (Figure 1.5) (Wang et al. 2006a; Haince et al. 2008). PARP-1 also physically and functionally interacts with two phosphatidyl inositol 3-like protein kinases ATM (involved in HR) and DNA-PKcs (involved in NHEJ), which are crucial for DSB signaling (Ruscetti et al. 1998; Aguilar Quesada et al. 2007; Haince et al.

2007). The precise mechanism by which PARP-1 participates in DSB repair has to be elucidated. However, it was suggested that PARP-1 serves as a general DNA damage detecting molecule, which potentially mediates a switch between the NHEJ and the HR pathways (Beneke and Bürkle 2007; Shrivastav et al. 2008). Consistent with this, PARP-1 was shown to function in a NHEJ backup pathway (Audebert et al. 2004; Wang et al. 2006a), whereas several reports demonstrated an anti-recombinogenic activity of PARP-1 (Waldman and Waldman

1991; Semionov et al. 2003; Dominguez-Bendala et al. 2006). The anti-recombinogenic activity of PARP-1 is supported by the finding that homologous recombination, leading to sister chromatid exchange (Sonoda et al. 1999), is inhibited by overexpression of PARP-1 (Meyer et al. 2000) and increased by inhibition of PARP activity (Dominguez et al. 2000).

1.1.2.5.2.4 PARP-1, Telomeric Maintenance, and the WRN Protein

Telomeres constitute repetitive sequences at the end of the chromosomes and function as a buffer to prevent loss of coding sequences during DNA replication. They are capped by a protein complex known as shelterin, which tightly regulates the telomeric structure by an interplay with several DNA repair proteins and the telomere-elongating reverse transcriptase, telomerase (Hoeijmakers 2001; de Lange 2005).

To date, the role of PARP-1 in the regulation of telomere length in vivo is controversial, ranging from PARP-1-deficiency not affecting telomere lengths (Samper et al. 2001) to a substantial overall loss of telomeric DNA by 30% in the first generation of Parp-1 knock-out mice (d'Adda di Fagagna et al. 1999). PARP-1 interacts with and modifies the telomeric repeat binding factor 2 (TRF2), a constituent of the shelterin complex (Figure 1.5). The poly(ADP-ribosyl)ation of TRF2 also affects its binding to telomeric DNA (O'Connor et al. 2004; Gomez et al. 2006). Gomez et al. reported that PARP-1 is dispensable for the capping of normal telomeres, but is specifically recruited to eroded telomeres, where it might help to protect chromosomes against end to end fusions and genomic instability (Gomez et al. 2006).

Another protein, involved in telomere regulation and general genome maintenance, is the RecQ helicase WRN. In addition to its helicase activity, WRN exhibits a 3‟-5‟ exonuclease activity. Patients with the rare autosomal recessive disorder Werner syndrome, in which the WRN gene is mutated, display genomic instability and telomere shortening on the cellular and a premature aging syndrome on the organismal level. The premature aging phenotype of these patients appears to be at least partly dependent on telomere length, since human symptoms were only recapitulated in mice with short telomeres, i.e. WRN/telomerase double knock-out mice (Brosh and Bohr 2007). [NB. Mice usually exhibit considerably longer telomeres (~40 kb) than humans (5-15 kb)]. PARP-1 modulates WRN exonuclease and helicase activity, presumably via physical interaction (Figure 1.5) (Adelfalk et al. 2003; von Kobbe et al. 2004). It was shown that functional WRN is required for PARP-1-dependent poly(ADP-ribosyl)ation, as this process is reduced in WRN-deficient cells (von Kobbe et al. 2003). Genetic cooperation between PARP-1 and WRN was also demonstrated in vivo, because mice with mutations in both proteins display higher rates of chromatid breaks, chromosomal rearrangements and cancer (Lebel et al.

2003). Since PARP-1 and WRN share many interaction partners (e.g. p53, DNA-PK, TRF2) and both proteins participate in other DNA repair pathways such as BER and NHEJ, they probably synergistically collaborate to maintain overall genomic stability.

1.1.2.5.2.5 PARP-1 and Mitotic Regulation

Proper mitotic regulation is crucial to ensure genomic integrity (Scholey et al. 2003). During mitosis, the spindle pole formation requires the centrosome, while the centromere is the chromosomal region that organizes the kinetochore, thus enabling the attachment of the mitotic spindle microtubules.

Haploinsufficiency for PARP-1 was recently shown to be related to centrosome duplication and chromosomal instability (Kanai et al. 2007b). Consistent with this, PARP-1 localizes to the centrosome (Kanai et al. 2000; Kanai et al. 2003). Moreover, PARP-1 and PARP-2 are present at centromeres and interact with the constitutive centromere proteins Cenpa, Cenpb and the spindle check point protein Bub3 (Figure 1.5) (Saxena et al. 2002a; Saxena et al. 2002b). The role of PARP-1 in spindle regulation is also supported by another study with Xenopus laevis egg extracts showing that poly(ADP-ribose) is a component of the mitotic spindle and is required for its assembly and function (Chang et al. 2004).

In summary, the physical and functional relationship of PARP-1 to the centrosome and the centromere links DNA damage surveillance to the mitotic spindle checkpoint.

1.1.2.5.2.6 PARP-1, P53, and Cell Cycle Control

Severe DNA damage or mitotic misregulation can cause genomic instability, leading to tumor formation. A complex cellular security network has evolved to counteract carcinogenesis. This signaling network can stop the cell cycle at different stages, thereby either leading to the eradication or neutralization of the cell by apoptosis or senescence, respectively, or to DNA damage repair.

A key player in this process is the tumor suppressor protein p53 (Aylon and Oren 2007). It was shown that more than half of the human tumors bear mutations in one or both alleles of p53 (Weinberg 2007). P53 acts as a transcription factor that physically interacts with PARP-1 (Figure 1.5) (Kumari et al. 1998). Consistent with the role of PARP-1 and p53 as caretakers and guardians of the genome, respectively, in vivo PARP-1 and p53 synergistically cooperate in telomere and chromosomal maintenance as well as in tumor suppression (Beneke and Moroy 2001; Tong et al. 2001a; Tong et al. 2001b; Tong et al. 2003; Wesierska-Gadek et al. 2005;

Tong et al. 2006). On a cellular level, many functional interactions between PARP-1 and p53 during DNA damage response and apoptosis were reported, such as delayed p53 transactivation potential in PARP-1-deficient cells (Kumari et al. 1998; Wang et al. 1998; Valenzuela et al.

2002; Ishizuka et al. 2003; Wieler et al. 2003; Bürkle 2006). On the other hand, poly(ADP-ribosyl)ation of p53 inhibits its binding affinity to its transcriptional consensus sequence, indicating complex and multifaceted regulatory mechanism (Mendoza-Alvarez and Alvarez-Gonzalez 2001; Simbulan-Rosenthal et al. 2001). Kanai et al. suggested a mechanism of PARP-1-dependent regulation of p53 activity: According to this model poly(ADP-ribosyl)ation

induces structural changes in p53 that mask its nuclear export sequence, resulting in an accumulation of p53 in the nucleus where it exerts its transactivational functions (Kanai et al.

2007a).

PARP-1 is also associated with a p53 downstream target, the cell cycle-dependent kinase inhibitor p21, which cooperates with PARP-1 in regulating the functions of the proliferating nuclear antigen (PCNA) during DNA replication and repair (Figure 1.5) (Frouin et al. 2003).

1.1.2.5.3 PARP-1, NF-κB, and Transcription

Previously, PARP-1 was identified as a transcription factor (TFII-C), which is associated with RNA polymerase II-dependent transcription (Slattery et al. 1983). This view was supported by more recent studies, suggesting that PARP-1 regulates RNA polymerase II-dependent transcription through the poly(ADP-ribosyl)ation of transcription factors, which may consequently alter their DNA binding affinity (Meisterernst et al. 1997; Oei et al. 1998).

Consistently, PARP-1 deficiency alters expression of genes involved in cell cycle progression, DNA replication, oxidative stress, cancer initiation and aging (Simbulan-Rosenthal et al. 2000;

Deschenes et al. 2005). Two modes of PARP-1-dependent transcriptional regulation were postulated: (i) a histone modifying enzymatic activity that can modulate chromatin architecture and (ii) an enhancer/promoter binding cofactor activity, which depends either on the mere presence of PARP-1 or on PARP-1 enzymatic activity (Kraus and Lis 2003).

The mechanism of the first mode of action, discussed in section 1.1.2.5.1, was impressively illustrated in a drosophila study by Tulin and Spradling. These authors demonstrated that PARP-1 is crucial for puff formation in Drosophila melanogaster giant polytene chromosomes.

Puff formation arises from local loosening of the chromatin structure and is associated with actively transcribed regions (Tulin and Spradling 2003). Recent findings indicate that transcription is also regulated by poly(ADP-ribosyl)ation of chromatin components other than histones, e.g. the DEK protein. DEK, a major component of mammalian chromatin with pleiotropic functions in DNA- and RNA-dependent processes, is a target of poly(ADP-ribosyl)ation, upon which DEK is released from chromatin to permit transcriptional initiation (Gamble and Fisher 2007; Kappes et al. 2008). Ju et al. provided interesting mechanistic evidence linking PARP-1-dependent initiation of transcription and its function in DNA binding and repair. According to their model, PARP-1 acts in concert with another binding partner, topoisomerase II. Topoisomerase II introduces a transient double strand break at the promoter which leads to PARP-1 binding and activation. The subsequent rapid but transient poly(ADP-ribosyl)ation triggers chromatin relaxation and initiation of transcription. PARP-1 activation is also necessary for the exchange of histone H1 with HMGB proteins, which are chromatin-associated proteins that bend DNA and recruit transcription factors to their DNA targets (Ju et al. 2006).

With respect to the second mode of action, i.e. the enhancer/promoter cofactor activity of PARP-1, it was shown that PARP-1 can act as a transcriptional coactivator as well as a repressor. Also, both PARP-1 enzyme-activity-dependent and independent mechanism were suggested. Regulatory effects of PARP-1 on transcription were shown in the case of various transcription factors such as AP-1/2 (Kannan et al. 1999; Andreone et al. 2003), p53 (Simbulan-Rosenthal et al. 2001), B-Myb (Cervellera and Sala 2000), TEF-1/Max (Ju et al. 2004), SP1 (Zaniolo et al. 2007), YY-1 (Oei and Shi 2001), STATs (Goenka et al. 2007), NFAT (Olabisi et al. 2008; Valdor et al. 2008), Oct-1 (Nie et al. 1998), TCF4 (Idogawa et al. 2005; Idogawa et al.

2007), RAR (Pavri et al. 2005), RXR (Miyamoto et al. 1999), progesterone receptor (Sartorius et al. 2000), and NF-κB (Hassa and Hottiger 1999).

Maybe the best studied interaction is that of PARP-1 with the transcription factor nuclear factor kappa B (NF-κB, Figure 1.6). NF-κB is composed of dimeric combinations of Rel transcription factor family members. It is of particular importance in the regulation of gene expression in cells of the immune system and was linked to the cellular immune and inflammatory response (Bürkle 2006). The expression and activation pattern of PARP-1 and NF-κB is remarkably similar in various tissues. However, the strongest evidence for a direct role of PARP-1 in NF-κB-mediated transcription was given by the finding that expression of NF-κB-dependent pro-inflammatory mediators, such as TNF-α, IL-6, or iNOS is impaired in Parp-1 knock-out mice (section 1.1.2.6.1) (Oliver et al. 1999; Hassa and Hottiger 2002). It was shown that PARP-1 physically interacts with both major subunits, i.e. p65 and p50, of NF-κB and is required for NF-κB-dependent gene transcription (Hassa and Hottiger 1999). Importantly, neither the DNA binding nor the enzymatic activity of PARP-1 were necessary for full activation of NF-κB (Hassa et al. 2001). Recently, it was shown that PARP-1 is acetylated by the histone acetylase p300/CBP upon inflammatory stimuli, leading to a stronger association with NF-κB (Hassa et al. 2005). The functional interaction of PARP-1 and NF-κB provides an important link to the role of PARP-1 in cell death and disease as described in the following sections (Figure 1.6 and Figure 1.7).

1.1.2.5.4 PARP-1 and its Role in Cell Death

DNA damage is a potent trigger of cell death. Historically, two major mechanisms of mammalian cell death are distinguished, apoptosis and necrosis. Classical apoptosis is considered as the default pathway, where cell death occurs in a controlled manner resulting in the elimination of cells by macrophages without secondary damage of the surrounding cells. In contrast, necrosis is considered as an uncontrolled process, which leads to disruption of cells

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