InteractiveFly: GeneBrief

Poly-(ADP-ribose) polymerase: Biological Overview | References

Protein ADP ribosylation catalyzed by cellular poly(ADP-ribose) polymerases (PARPs) and tankyrase [an enzyme that modulates the activity of target proteins through poly(ADP-ribosyl)ation], modulates chromatin structure, telomere elongation, DNA repair, and the transcription of genes involved in stress resistance, hormone responses, and immunity. Using Drosophila genetic tools, the expression and function of poly(ADP-ribose) glycohydrolase (PARG), the primary enzyme responsible for degrading protein-bound ADP-ribose moieties, was characterize. Strongly increasing or decreasing PARG levels mimics the effects of Parp mutation, supporting PARG's postulated roles in vivo both in removing ADP-ribose adducts and in facilitating multiple activity cycles by individual PARP molecules. PARP is largely absent from euchromatin in PARG mutants, but accumulates in large nuclear bodies that may be involved in protein recycling. Reducing the level of either PARG or the silencing protein SIR2 weakens copia transcriptional repression. In the absence of PARG, SIR2 is mislocalized and hypermodified. It is proposed that PARP and PARG promote chromatin silencing at least in part by regulating the localization and function of SIR2 and possibly other nuclear proteins (Tulin, 2006).

ADP-ribose modification of nuclear proteins mediates DNA repair, gene transcription, telomere elongation, and chromatin structure (reviewed in Ziegler, 2001; Tulin, 2003b). Protein ADP-ribosylation levels are ultimately determined by the location and activity of poly(ADP-ribose) polymerase (PARP) and tankyrase enzymes that utilize NAD to add such residues, as well as poly(ADP-ribose) glycohyrolase (PARG) enzymes that remove them. Although a great deal has been learned about the biochemical properties of these enzymes in vitro, exactly how they function in vivo remains poorly known. Most of the time, the vast majority of PARP molecules are enzymatically inactive, unmodified, and thought to act only during brief bursts of activity. Damaged or altered DNA conformation, along with other uncharacterized signals, can cause nearby PARP molecules within small chromosome regions to dimerize and become transiently active before they are dissociated and shut off by automodification with long poly(ADP-ribose) chains (pADPr). Histones and other chromosomal proteins in the affected chromatin domain adopt a looser configuration, either by binding avidly to PARP-linked poly(ADP-ribose) polymers or by direct modification, thereby facilitating repair or gene activation (reviewed in Tulin, 2003b). When PARG eventually removes all the ADP-ribosyl groups from a PARP monomer, the cycle can repeat until the inducing conditions are no longer present. Other mechanisms of PARP action have been proposed as well, including some that do not require PARP enzymatic function (Tulin, 2002; Ju, 2004; Kim 2004; Tulin, 2006 and references therein).

Genetic analysis of this system is greatly facilitated in Drosophila, which contains a single Parp gene located in 3R heterochromatin that encodes an enzyme with the same domain structure as that of the major mammalian PARP1 protein (Hanai, 1998; Tulin, 2002). Drosophila also contains a single tankyrase gene (tankyrase) and a single gene (Parg) predicted to encode a PARG (Hanai, 2004). Parp mutations are lethal and drastically alter many aspects of developmental physiology (Tulin 2002; Tulin, 2003a). These include the ability to activate and maintain nucleoli, to form polytene chromosome puffs, and to activate genes located therein that respond to stress, infection, or steroid hormones (Tulin, 2006).

Heterochromatin forms in early embryonic cells and additional chromatin domains are silenced as individual cell types differentiate. The ability to compact heterochromatin and to silence specific gene regions also requires Parp. For example, the 30-50 normally quiescent genomic copies of the copia retrotransposon are overexpressed >50-fold in Parp mutants (Tulin 2002). Normally, copia transcription is suppressed by a chromatin-based mechanism related to gene silencing in other regions. Thus, in addition to its role as an activator, PARP contributes to the repression of at least some chromatin domains (Tulin, 2006).

The evolutionarily conserved silent information repressor protein 2 (SIR2; see Drosophila Sir2) protein contributes to heterochromatin formation through the action of its NAD-dependent histone deacetylase activity. NAD is cleaved in conjunction with removal of acetyl groups from the target, forming nicotinamide and O-acetyl-ADP-ribose. In addition, many SIR2 protein family members catalyze protein ADP ribosylation (Frye, 1999). Drosophila contains five genes related to yeast SIR2, but the Sir2 gene residing at 34A7 shows the highest level of conservation and exhibits NAD-dependent histone deacetylase activity. While nonessential, Sir2 participates in chromatin silencing (Tulin, 2006).

To better understand how poly(ADP)-ribose metabolism regulates chromatin activity, this study characterized the Drosophila Parg gene (see also Hanai, 2004). The findings reinforce previous evidence that PARP-catalyzed ADP ribosylation plays widespread roles in the nucleus, which are not limited to DNA repair. They support the view that PARP acts in vivo by undergoing bursts of activation limited by automodification and reversed by PARG action. In addition, PARG was found to control the localization of other nuclear proteins. In Parg mutants, SIR2 protein is mislocalized and hypermodified; endogenous copia retrotransposon expression is elevated, suggesting that chromatin silencing is compromised. These experiments further document important roles played by ADP-ribose modification in controlling chromatin structure and activity and suggest that some of these effects are mediated through SIR2 (Tulin, 2006).

A previous study (Hanai, 2004) found that Parg mutations are lethal or semilethal, and an effect of mutations on pADPr levels in neural tissue and on organismal life span was reported. However, the relationship between PARG and the roles played by PARP (Tulin 2002; Tulin, 2003a) remained unclear. This study has shown that reducing or increasing PARG causes phenotypic effects very similar to disrupting Parp on nucleolar function, chromatin structure, immunity, and heat-shock sensitivity. This shows that PARG acts in many common pathways with PARP, presumably by virtue of its enzymatic action on ADP-ribose groups (Tulin, 2006).

The phenotypic similarity observed among Parg disruption, PARG overproduction, and Parp mutation argues that protein ADP ribosylation, rather than a direct structural function, underlies many of the reported actions of PARP. Molecular studies of Parg mutants directly verified the predicted increase in ADP-ribose modification levels. In the absence of PARG, newly synthesized PARP molecules would still be expected to function until they became automodified. Thus, the strong phenotypic effects of Parg mutation imply that recycling of automodified PARP molecules is quantitatively important, at least locally. For example, the developmental delays that were observed prior to each molt are probably caused by the extra time needed to synthesize de novo enough new PARP to support molting gene expression. PARG overproduction would also be expected to interfere with PARP action. Poly(ADP-ribose) chains on automodified PARP might remain too short to function, and protein recycling that depends on the kinetics of poly(ADP-ribose) modification might be disrupted. In support of these interpretations, it was found that the phenotypic effects of PARG overproduction are completely suppressed by simultaneously producing extra PARP (Tulin, 2006).

Consequently, these studies strongly support the view that localized episodes of poly(ADP)-ribose modification under the control of PARP, and recycling of ADP-ribose-modified proteins under the control of PARG, play a major role in controlling chromatin structure, gene activity and nuclear function in vivo. However, it still remains unclear how PARP is incorporated into chromatin in an inactive state, how it becomes locally activated (except in the case of DNA damage), and to what extent other chromatin proteins in addition to PARP itself are important substrates for PARP and PARG enzymatic activities in vivo. Moreover, this work does not rule out the possibility that PARP also acts via other mechanisms, including some that do not require enzymatic function. Drosophila oocytes and early embryos contain an essential isoform, PARP-e, that lacks a catalytic domain (Tulin 2002). In vitro, human PARP-1 represses chromatin by binding to nucleosomes, displacing histone H1, and compacting its local architecture independently of PARP enzymatic activity (Kim, 2004). In addition, PARP may form a stable component of repressive chromatin complexes on target genes (Ju, 2004; Tulin, 2006 and references therein).

The phenotype of Parg disruption is not identical to Parp mutation, suggesting that PARG carries out some functions independently of PARP. In particular, PARP protein and Fibrillarin accumulate in large nucleoplasmic and peri-centromeric bodies in Parg mutant cells, in contrast to the Fibrillarin-rich cytoplasmic body seen in Parp mutants. It is suggested that these structures correspond to intermediates in a process that normally recycles ADP-ribose-modified proteins within the cell. In this respect, they are reminiscent of Cajal bodies, which have been postulated to serve as staging, storage, or assembly sites of factors involved in transcript production and processing. The normal rate of this recycling may be greater in regions of high gene activity. When ADP-ribose groups cannot be removed, the recycling process backs up, causing the observed breakdown of the nucleolus and loss of PARP from eukaryotic chromosome regions. The nucleolus might be particularly sensitive if ongoing ADP ribosylation is needed to maintain rDNA genes, which do not exhibit a normal nucleosomal organization, in an active state. The phenotypic differences between Parp and Parg mutants may result from different arrest points within a common recycling pathway or because PARG also reverses the action of other poly(ADP-ribose) polymerases in addition to PARP (Tulin, 2006).

PARP activation or PARG reduction might block SIR2 action simply by depleting cellular NAD pools (Zhang, 2003). However, PARG overexpression should not reduce NAD pools, and yet nucleolar structure was disrupted in animals with elevated PARG. Instead of acting via NAD, the observation that, in the absence of PARG, a higher-molecular-weight form of SIR2 accumulates in the cell cytoplasm suggests that SIR2 is modified by an ADP-ribose addition as part of its function. Many SIR2 protein family members themselves exhibit protein ADP-ribosylation activity (Frye, 1999; Furuyama, 2004), and mouse SIRT6, a predominantly nuclear protein, can direct its own mono-(ADP) ribosylation (Liszt, 2005). PARG may be needed to remove ADP-ribose groups from SIR2 that are added by these or other mechanisms that are independent of PARP (Tulin, 2006).

Taken together, these experiments suggest a model in which PARP, PARG, and SIR2 cooperate to silence specific chromosomal domains. It is proposed that activation and ADP ribosylation of PARP molecules (and possibly other local chromatin proteins) loosen chromatin early in the silencing process and that this facilitates SIR2 access to acetylated histone tails. In some cases this process would transiently strip the target chromatin proteins off the affected region and transfer them in an organized fashion to the branched ADP-ribose polymers on auto-inactivated PARP molecules within the immediately adjacent nucleoplasm. Here the ADPr/chromatin complex would encounter PARG and SIR2, possibly in conjunction with other proteins involved in chromatin remodeling (Furuyama, 2004). SIR2 molecules would undergo autoADP-ribosylation and deacetylate histones such as H4 within the complex, while PARG begins to cleave their ADP-ribose moieties. Since the ADPr tails shorten, the chromatin proteins would be driven to reassemble onto their former chromosome region. PARG action on SIR2 might also help coordinate these events (Tulin, 2006).

When Parp is mutated or when PARG activity becomes too high or too low, chromatin activation and silencing would be drastically disrupted. Without PARP or in the presence of excess PARG, chromatin proteins would fail to loosen and become accessible to modification. The state of chromatin would become 'frozen' at whatever state it had reached when the deficiency became acute (i.e., when maternal PARP is depleted in the case of a zygotic Parp mutant, or when expressed PARG reaches a critical level). When PARG levels are too low, in contrast, excess levels of ADP-ribose-modified SIR2 would build up, driving it into the cytoplasm. Following a single activation, PARP molecules would be trapped in the inactive automodified state. Large amounts of pADPr would accumulate, shunting chromatin proteins into remodeling complexes that cannot break down. It is now possible to look forward to obtaining a more detailed understanding of these events using the genetic tools available for the study of chromatin organization in Drosophila (Tulin, 2006).

Nucleosomal core histones mediate dynamic regulation of Poly(ADP-ribose) polymerase 1 protein binding to chromatin and induction of its enzymatic activity

Poly(ADP-ribose) polymerase 1 protein (PARP1) mediates chromatin loosening and activates the transcription of inducible genes, but the mechanism of PARP1 regulation in chromatin is poorly understood. This study found that Drosophila PARP1 interaction with chromatin is dynamic and that PARP1 is exchanged continuously between chromatin and nucleoplasm, as well as between chromatin domains. Specifically, the PARP1 protein preferentially interacts with nucleosomal particles, and although the nucleosomal linker DNA is not necessary for this interaction, the core histones, H3 and H4, are critical for PARP1 binding. Histones H3 and H4 interact preferentially with the C-terminal portion of PARP1 protein, and the N-terminal domain of PARP1 negatively regulates these interactions. Finally, it was found that interaction with the N-terminal tail of the H4 histone triggers PARP1 enzymatic activity. Therefore, the data collectively suggests a model in which both the regulation of PARP1 protein binding to chromatin and the enzymatic activation of PARP1 protein depend on the dynamics of nucleosomal core histone mediation (Pinnola, 2007).

This paper provides the first insight into the nature of the association of the PARP1 protein with chromatin in vivo and in vitro. The dynamics between free and chromatin-bound PARP1 protein were characterized, and an additional mechanism for these interactions is suggested. It was also demonstrated that PARP1 associates with chromatin on a monucleosomal level in vivo. More specifically, H3 and H4 are preferential binding sites for the C-terminal domain of PARP1 and that DNA is not required for this association in vitro. Histone H4 works as a strong DNA-independent activator of pADPr enzymatic reaction, whereas other histones (especially H2A) inhibit H4-dependent PARP1 activation (Pinnola, 2007).

PARP1 protein is exchanged rapidly between chromatin regions in the nucleus. No difference was detected between the recovery rate of enzymatically inactive PARPe-EGFP protein and active PARP1-DsRed protein isoforms. Therefore, it is proposed that PARP1 enzymatic activity is not required for steady-state dynamics. However, PARP1 inactivation followed by due automodification of PARP1 molecules has been shown to be critical for PARP1 protein removal from chromatin (Mendoza-Alvarez, 1993). The existence of two distinct mechanisms controlling PARP1 interaction with chromatin were detected as a result of sucrose gradient purification experiments. That is, unmodified PARP1 molecules co-purifies with nucleosomes, as well as other fractions (Complex I), whereas PARP1 molecules modified with pADPr were segregated to a separate fraction (Complex II). Based on this finding, it is concluded that, indeed, two distinct mechanisms conjoin to control PARP1 molecule interaction with chromatin. One involves a protein-equilibrated binding via association-dissociation, and the other involves irreversible removal of PARP1 from chromatin after automodification. Based on an accepted model, the existence of Complexes I and II was expectedPARP1 protein is associated with chromatin in its inactive state (Complex I), and upon activation it becomes automodified, loses contact with chromatin, and establishes interactions with pADPr-binding proteins (Complex II). Interestingly, the fraction with Complex II also contains a significant amount of unmodified PARP1. This may suggest that there is a nucleoplasmic pool of unmodified PARP1 that can reversibly bind to pADPr (Pinnola, 2007).

A three step model is presented for the regulation of PARP1 protein enzymatic activity in chromatin. Step 1: PARP1 protein is broadly distributed in chromatin because of interaction with core histones in the context of nucleosome. PARP1 is inactive in this state because of inhibitory effect of histone H2A. Step 2: genotoxic stress-dependent PARP1 activation. The N-terminal domain of PARP1 protein serves as a sensor of the double-stranded breaks or nicks in genomic DNA. Upon binding of damaged DNA, it mediates conformational changes, which leads to disruption of interaction with histones and consequently to the activation of PARP1 enzymatic reaction. Step 3: DNA-independent PARP1 activation. Developmental or environmental signals induces local changes in the "histone modification core" and subsequently expose the N-terminal tail of histone H4 and/or hide histone H2A followed by H4-dependent PARP1 activation (Pinnola, 2007).

Similar to H1, PARP1 controls the establishment of silenced chromatin (Tulin, 2002). Recently, it has been shown that PARP1 and H1 work independently. Moreover, they antagonize each other in chromatin (Kim, 2004). This antagonistic interaction strongly suggests competition for the same binding sites. The site of linker histone binding is known to be the linker DNA in the context of nucleosomal array. Unlike H1, linker DNA is not crucial for PARP protein binding. This, in turn, suggests that if H1 and PARP compete for binding sites, they recognize different but overlapping, epitopes (Pinnola, 2007).

The ability of PARP1 to bind chromatin via nicks in double-stranded DNA, as well as noncanonical DNA structures, has been demonstrated in vitro (Huang, 2004). Still, the broad PARP1 localization in chromatin in vivo suggests an alternative mechanism for PARP1 protein binding. Histones H2A and H2B have been reported as preferential targets for PARP1 binding in vitro (Buki, 1995) and for enzymatic modification by PARP1. In the current experiments, unmodified PARP1 protein always co-purified with core histones, even after DNA digestion to mononucleosomes. It was also found that the C terminus of PARP1 preferentially binds histones H3 and H4 of histone octamers lacking DNA. The PARP1 C terminus contains the catalytic domain and the sequence required for homodimerization and thus activation. PARP1 C terminus binding to H3/H4 may serve to sequester the domains in PARP1 that are required for activation, and this could account for the broad localization of PARP1 in chromatin. Histone H4 activates, whereas histone H2A completely inhibits, PARP1 protein. These findings support the conclusion that the PARP1 protein is generally silent (enzymatically inactive) in chromatin, although a number of developmental and environmental stimuli could still activate it at specific loci. This activation is required for chromatin decondensation and transcriptional activation in these loci. PARP1 activation always correlates with changes of local histone modification [e.g. phosphorylation of histone H3 co-localized with pADPr in Drosophila puffs (Tulin, 2003b)]. Therefore, it is hypothesized that changes in histone modification code promote conformational alteration of nucleosomes and therefore expose (or hide) specific domains of histones, which activate (or inhibit) PARP1 (Pinnola, 2007).

Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster

Poly(ADP-ribosyl)ation has been suggested to be involved in regulation of DNA repair, transcription, centrosome duplication, and chromosome stability. However, the regulation of degradation of poly(ADP-ribose) and its significance are not well understood. This study reports a loss-of-function mutant Drosophila with regard to poly(ADP-ribose) glycohydrolase, a major hydrolyzing enzyme of poly(ADP-ribose). The mutant lacks the conserved catalytic domain of poly(ADP-ribose) glycohydrolase, and exhibits lethality in the larval stages at the normal development temperature of 25 degrees C. However, one-fourth of the mutants progress to the adult stage at 29 degrees C but showed progressive neurodegeneration with reduced locomotor activity and a short lifespan. In association with this, extensive accumulation of poly(ADP-ribose) could be detected in the central nervous system. These results suggest that poly(ADP-ribose) metabolism is required for maintenance of the normal function of neuronal cells. The phenotypes observed in the parg mutant might be useful to understand neurodegenerative conditions such as the Alzheimer's and Parkinson's diseases that are caused by abnormal accumulation of substances in nervous tissue (Hanai, 2004. Full text of article).

Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci

Steroid response and stress-activated genes such as hsp70 undergo puffing in Drosophila larval salivary glands, a local loosening of polytene chromatin structure associated with gene induction. This study shows that ouffs acquire elevated levels of adenosine diphosphate (ADP)-ribose modified proteins, and poly(ADP)-ribose polymerase (PARP) is required to produce normal-sized puffs and normal amounts of Hsp70 after heat exposure. It is proposed that chromosomal PARP molecules become activated by developmental or environmental cues and strip nearby chromatin proteins off DNA to generate a puff. Such local loosening may facilitate transcription and may transiently make protein complexes more accessible to modification, promoting chromatin remodeling during development (Tulin, 2003a).

Cells within developing multicellular eukaryotes build complex tissue-specific chromatin architectures to express certain genes and silence others. The enzyme PARP is thought to play a critically important role in preserving differentiated chromatin during DNA repair. The protein's zinc fingers specifically recognize DNA damage, and PARP activity increases strongly upon binding to such sites. The activated enzyme modifies nearby chromatin proteins with ADP-ribose moieties, disrupting their macromolecular complexes, and causing the affected chromatin to decondense. The newly repaired region returns to a normal state after PARP down-regulates its own activity via automodification, and the chromatin proteins, freed of ADP-ribose groups by a specific glycohydrolase, reassemble. Recent genetic studies with Drosophila melanogaster show that PARP is an essential gene required to organize chromatin throughout the life cycle. However, the relation between PARP action during repair and during development remains unclear (Tulin, 2003a).

Drosophila chromatin normally undergoes many programmed changes that could be mediated by PARP. In particular, chromatin alterations manifest as salivary gland polytene chromosome puffs occur at the sites of ecdysone response genes before molting. To look for specific loci where PARP may act, the levels of PARP protein, ADP-ribosylation activity, and ADP-ribose polymers were surveyed during embryonic and larval development. Epitope-tagged PARP protein is widely distributed throughout the euchromatin of both diploid and polytene Drosophila nuclei. However, the activity state of these PARP molecules varies widely because injected biotinylated nicotinamide adenine dinucleotide (NAD), the source of ADP-ribose adducts, differentially labels only a limited number of polytene chromosome regions, and this labeling is blocked by the PARP inhibitor 3-aminobenzidine (3-AB). ADP-ribose polymers detected with a specific antibody are also found only within particular chromosome bands, including early ecdysone puff loci. For example, in the region surrounding the 75A-75B puffs, PARP protein is widespread, but high levels of poly(ADP-ribose) appear only at the time of puffing. These observations suggest that PARP becomes active during puff induction and modifies local proteins, leading to an accumulation of ADP-ribose moieties. PARP activation in ecdysone puffs may be functionally important because Parp mutant larvae frequently arrest at ecdysis (Tulin, 2003a).

To determine whether puff formation requires PARP, stress-induced puffs were studied. A heat shock strongly induces loci containing stress response genes to puff, including the clustered genes encoding Hsp70 chaperone at 87A and 87C. Before heat shock, PARP is widespread along polytene chromosomes, but little poly(ADP-ribose) is present at the 87A, 87C, and surrounding loci. In contrast, after a brief heat shock, ADP-ribose polymers accumulate differentially throughout the large, newly formed puffs. After 25 min of recovery, the amount of poly(ADP)-ribose begins to fall, and the puff itself begins to regress. These observations reveal a strong correlation between ADP-ribose accumulation and puffing (Tulin, 2003a).

To determine if the Parp gene encodes the enzyme responsible for elevating ADP-ribose levels during puffing and if this increase is functionally important, it was asked whether heat shock-induced puffs arise normally in Parp mutant larvae. Parp-defective animals die during the second instar, when salivary gland polytene chromosomes are too small to analyze cytologically. However, it was found that puffs in these animals can be quantitated by in situ hybridization with an hsp70 probe. The average size of the 87A and 87C puffs is reduced threefold in Parp mutant larvae. Complete loss of PARP likely has an even bigger effect, as the mutant animals continue to express low levels of PARP. Consistent with this expectation, puff formation at 87A and 87C was completely blocked in wild-type third instar larvae that were fed 3-AB for 1 hour, and these larvae failed to recover from heat shock. Western blots showed that 5 to 10 times less Hsp70 protein was induced by heat shock in mutant compared with wild-type larvae. Thus, PARP is needed to form normal heat shock puffs, to express normal levels of puff-encoded proteins, and to normally resist the deleterious effects of heat stress (Tulin, 2003a).

The association of PARP activity with developmental puffs and its requirement for heat shock-induced puffs suggests that some genes undergo chromatin loosening during induction. Immune response genes may belong to this group because Parp1 knockout mice display dramatic immune defects and cannot induce genes controlled by nuclear factor kappa B (NF-kappa B) transcription factors (Kameoka, 2000). Parp mutant Drosophila larvae frequently develop intracellular bacterial infections, a condition rarely observed in wild type. Spontaneous infection correlates with low PARP levels, because in larval tissues retaining variable amounts of PARP only cells with little or no PARP function become infected. Furthermore, Parp mutant larvae are sensitive to experimental infection. Ninety-five percent died after a dose of injected Escherichia coli bacteria that killed less than 7% of size-matched wild-type controls. Thus, like Parp1-deficient mice, Drosophila lacking normal PARP levels display immune defects (Tulin, 2003a).

Inducible innate immunity genes constitute a major resistance mechanism to microbial infection in insects such as Drosophila. The rapid production of antibacterial proteins by these genes is controlled by NF-kappa B-related transcription factors. The ability of wild-type and Parp mutant animals to induce two innate immunity genes, Diptericine and Drosomycin, was compared using Western analysis of Diptericine-lacZ and Drosomycin-GFP (where GFP is green fluorescent protein) reporter genes. Both genes are strongly induced in wild-type, 20 to 40 hours after bacterial challenge, but protein levels increased little if at all in Parp mutant animals under the same conditions. These experiments argue that PARP's role in NF-kappa B-dependent immune response gene expression has been conserved during evolution. These observations indicate that PARP may act on the chromatin organization of NF-kappa B target loci (Tulin, 2003a).

PARP's proposed action during DNA repair suggests a model for its role during puffing. Signals other than DNA lesions, including steroid hormones, stress, and infection, may activate PARP molecules at specific chromosome sites. After modification with poly(ADP-ribose), local chromatin proteins, transcription factors, and PARP molecules themselves are proposed to dissociate from the DNA and/or from pre-existing protein complexes, and to complex with nearby branched ADP-ribose polymers. While in this expanded state, the altered chromosome domain may be transcribed and its chromatin constituents modified. Subsequently, the ADP-ribose would be cleaved, allowing chromatin to reform in an unchanged or altered configuration as dictated by the modified constituents (Tulin, 2003a).

These results help clarify the biological importance of puffs, which has remained unclear because under certain conditions puffing and gene expression are separable. Formation of an expanded chromatin state, the puff, would require PARP. However, only the configuration of factors pre-existing at the target site would determine whether chromatin loosening leads to gene activation. The experiments also suggest a specific molecular mechanism for puffing. Nucleosomes in puffs lose their regular arrangement, and hsp70 region genomic DNA becomes greatly extended. Nucleosomal and linker histones may be targets of ADP ribosylation by activated PARP molecules, causing chromatin in puffed regions to expand (Tulin, 2003a).

These experiments also suggest that genes located within repressed chromatin could be exposed and made more susceptible to reprogramming by intentionally activating nearby chromosomal PARP molecules. Understanding how PARP is activated within normal, undamaged chromatin will advance knowledge of developmental gene regulation and facilitate the development of methods to experimentally reprogram genes (Tulin, 2003a).

The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development

Poly(ADP-ribose) polymerase (PARP) is a major NAD-dependent modifying enzyme that mediates important steps in DNA repair, transcription, and apoptosis, but its role during development is poorly understood. A single Drosophila Parp gene spans more than 150 kb of transposon-rich centromeric heterochromatin and produces several differentially spliced transcripts, including a novel isoform, PARP-e, predicted to encode a protein lacking enzymatic activity. An insertion mutation near the upstream promoter for Parp-e disrupts all Parp expression. Heterochromatic but not euchromatic sequences become hypersensitive to micrococcal nuclease, nucleoli fail to form, and transcript levels of the copia retrotransposon are elevated more than 50-fold; the variegated expression of certain transgenes is dominantly enhanced. Larval lethality can be rescued and PARP activity restored by expressing a cDNA encoding PARP-e. It is proposed that PARP-e autoregulates Parp transcription by influencing the chromatin structure of its heterochromatic environment. The results indicate that Parp plays a fundamental role organizing the structure of Drosophila chromatin (Tulin, 2002. Full text of article).

Genomic organization of Drosophila poly(ADP-ribose) polymerase and distribution of its mRNA during development

Poly(ADP-ribosyl)ation of proteins catalyzed by poly(ADP-ribose) polymerase (PARP) modulates several biological activities. However, little is known about the role of PARP in developmental process. This study reports the organization of the Drosophila PARP gene and the expression patterns during Drosophila development. The Drosophila PARP gene is a single copy gene mapped at 81F and is composed of six exons. Organization of exons corresponds to the functional domains of PARP. The DNA-binding domain is encoded by exons 1, 2, 3, and 4. The auto-modification domain is encoded by exon 5, and the catalytic domain is in exon 6. The promoter region of the PARP gene contains a putative TATA box and CCAAT box unlike human PARP. Expression of the PARP gene is at high levels in embryos at 0-6 h after egg laying and gradually decreases until 8 h. PARP mRNA increases again at 8-12 h and is observed in pupae and adult flies but not in larvae. In situ mRNA hybridization of embryos revealed large amount of PARP mRNA observed homogeneously except the pole cells at the early stage of embryos, possibly due to presence of the maternal mRNA for PARP, and that decreases gradually until the stage 12 in which stage PARP mRNA localizes in anal plates. At late stage of embryogenesis PARP mRNA is localized in cells along the CNS (Hanai, 1998. Full text of article).

Search PubMed for articles about Drosophila Parp

Buki, K. G., Bauer, P. I., Hakam, A. and Kun, E. (1995). Identification of domains of poly(ADP-ribose) polymerase for protein binding and self-association. J. Biol. Chem. 270: 3370-3377. PubMed Citation: 7852424

Frye, R. A. (1999). Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP-ribosyltransferase activity. Biochem. Biophys. Res. Commun. 260: 273-279. PubMed Citation: 10381378

Furuyama, T., Banerjee, R., Breen, T. R. and Harte, P. J. (2004). SIR2 is required for Polycomb silencing and is associated with an E(Z) histone methyltransferase complex. Curr. Biol. 14: 1812-1821. PubMed Citation: 15498488

Hanai, M., et al. (1998). Genomic organization of Drosophila poly(ADP-ribose) polymerase and distribution of its mRNA during development. J. Biol. Chem. 273: 11881-11886. PubMed Citation: 9565614

Hanai, S., et al. (2004). Loss of poly(ADP-ribose) glycohydrolase causes progressive neurodegeneration in Drosophila melanogaster. Proc. Natl. Acad. Sci. 101: 82-86. PubMed Citation: 14676324

Huang, K., Tidyman, W. E., Le, K. U., Kirsten, E., Kun, E. and Ordahl, C. P. (2004). Analysis of nucleotide sequence-dependent DNA binding of poly(ADP-ribose) polymerase in a purified system. Biochemistry 43: 217-223. PubMed Citation: 14705948

Ju, B.-J., et al. (2004). Activating the PARP-1 sensor component of the Groucho/TLE1 corepressor complex mediates a CaMKinase II-dependent neurogenic gene activation pathway. Cell 119: 815-829. PubMed Citation: 15607978

Kameoka, M., et al. (2000). Evidence for regulation of NF-kappaB by poly(ADP-ribose) polymerase. Biochem. J. 346: 641-9. PubMed Citation: 10698690

Kim, M. Y., et al. (2004). NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1. Cell 119: 803-814. PubMed Citation: 15607977

Liszt, G., et al. (2005) Mouse Sir2 homolog SIRT6 is a nuclear ADP-ribosyltransferase. J. Biol. Chem. 280: 21313-21320. PubMed Citation: 15795229

Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (1993). Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular. J. Biol. Chem. 268: 22575-22580. PubMed Citation: 8226768

Pinnola, A., Naumova, N., Shah, M. and Tulin, A. V. (2007). Nucleosomal core histones mediate dynamic regulation of Poly(ADP-ribose) polymerase 1 protein binding to chromatin and induction of its enzymatic activity. J. Biol. Chem. 282(44): 32511-32519. PubMed Citation: 17827147

Tulin, A., Stewart, D. and Spradling, A. C. (2002). The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev. 16: 2108-2119. PubMed Citation: 12183365

Tulin, A. and Spradling, A. (2003a). Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299(5606): 560-2. PubMed Citation: 12543974

Tulin, A., Chinenovy, Y. and Spradling, A. C. (2003b). Regulation of chromatin structure and gene activity by poly(ADP-ribose) polymerases. Curr. Top. Dev. Biol. 56: 55-83. PubMed Citation: 14584726

Tulin, A., Naumova, N. M., Menon, A. K. and Spradling, A. C. (2006). Drosophila poly(ADP-ribose) glycohydrolase mediates chromatin structure and SIR2-dependent silencing. Genetics 172(1): 363-71. PubMed Citation: 16219773

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date revised: 9 February 2009

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