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).

Drosophila histone H2A variant (H2Av) controls poly(ADP-ribose) polymerase 1 (PARP1) activation in chromatin

According to the histone code hypothesis, histone variants and modified histones provide binding sites for proteins that change the chromatin state to either active or repressed. This study identified histone variants that regulate the targeting and enzymatic activity of poly(ADP-ribose) polymerase 1 (PARP1), a chromatin regulator in higher eukaryotes. PARP1 is targeted to chromatin by association with the histone H2A variant (H2Av)--the Drosophila homolog of the mammalian histone H2A variants H2Az/H2Ax--and subsequent phosphorylation of H2Av leads to PARP1 activation. This two-step mechanism of PARP1 activation controls transcription at specific loci in a signal-dependent manner. This study establishes the mechanism through which histone variants and changes in the histone modification code control chromatin-directed PARP1 activity and the transcriptional activation of target genes (Kotova, 2011).

The histone code hypothesis has long been accepted in the study of epigenetics, but there never has been a clear demonstration of the direct activation of an effector protein in response to changes in the histone environment. This study found that PARP1 activation and PARP1- mediated transcription depend on the regulation of a nucleosome's microenvironment, a mechanism that involves the phosphorylation of a histone variant (see Model of PARP1 protein regulation by histone H2Av variant and H2Av phosphorylation). This result supports the histone code hypothesis, and the underlying work also reveals a mechanism for PARP1 activation that is functionally important for the regulation of transcription, response to genotoxic stress, and silencing (Kotova, 2011).

Histone variant H2Av in Drosophila - and its homologs in Arabidopsis and Saccharomyces cerevisiae - localize in the promoter region of a subset of genes. These findings demonstrate that this localization is functionally significant. Specifically, H2Av is involved in the positioning and activation of the PARP1 protein. Nucleosomes containing H2Av form high-affinity sites at which the effector protein PARP1 binds with specific promoters. Thereafter, phosphorylation of H2Av alters the interaction of PARP1 with the nucleosomal histone H4, an event which, in turn, activates PARP1, leading to chromatin opening and facilitating transcription (Tulin, 2003a; Petesch, 2008; Kim, 2004). Therefore, taken as a whole, the results of this study show that, by recruiting PARP1 protein, H2Av controls the chromatin state as well as transcription activation and genotoxic stress response (Kotova, 2011).

Having established that H2Av controls the PARP1 function both in vivo and in vitro, the causal underlying mechanism was examined. Nucleosomes containing H2Av have been reported previously to have a 'more open' and stable conformation, suggesting that the presence of H2Av may increase access to other core histones, i.e., those hidden in H2A-containing nucleosomes. Among the histones, PARP1 protein preferentially interacts with H3/H4 tetramers, possibly explaining the enrichment of PARP1 in the presence of H2Av-containing nucleosomes in vivo, as reported in this study. In other words, chromatin in 'more open; H2Av nucleosomes, with a high level of H3/H4 exposure, has a greater affinity for binding with PARP1 than does unexposed chromatin. Moreover, it was found that interaction with the N-terminal tail of the histone H4 triggers PARP1 protein activation. The SQ domain of histone H2Av, the phosphorylation of which controls PARP1 activation in vivo, as reported in this study, is positioned in close proximity to the N-tail of H4 in the nucleosome. Therefore, it is proposed that the histone-replacement machinery positions H2Av within the promoter region of specific genes, thereby creating nucleosomes with an 'open' configuration. Within these nucleosomes, exposed H3/H4 histones bind PARP1 protein and properly determine its localization in promoters. Phosphorylation of the H2Av C terminus then leads to exposure of the H4 histone N-tail, promoting its interaction with PARP1, and the activation of the PARP1 protein (Kotova, 2011).

Although these results establish a direct connection between PARP1, H2Av-containing nucleosomes, H2Av phosphorylation, and pADPr, a possible role for H2Av phosphorylation itself cannot be excluded, in regulating the activity of the PARP1 protein on a higher level of chromatin organization. This possibility is suggested by the observed difference between the in vivo and in vitro results. Although phosphorylation of H2Av was required to elicit PARP1 activation in vivo, purified nucleosomes containing either H2A or H2Av were able to elicit PARP1 activation in vitro. Mimicking the phosphorylation of H2Av (as in H2AvSE) resulted in a 26% increase in the activation of PARP1 in vitro. These observations suggest that although phospho-H2Av may act directly on PARP1, it also may mediate changes in the higher-order chromatin microenvironment (which could not be reproduced in vitro), leading to the disruption of PARP1 interaction with inhibitors and/or the interaction of PARP1 with activating epitopes in the context of the local chromatin. Alternatively, H2Av phosphorylation may be involved in a 'system restart' in vivo; i.e., phosphorylation of H2Av has been linked to the replacement of this histone in the local chromatin. Consequently, multiple repeated acts of transcriptional initiation, and therefore multiple acts of H2Av phosphorylation, may be required, for example, during heat-shock gene expression. Thus, in the absence of H2Av phosphorylation, H2Av replacement will be blocked, and transcriptional restart will be arrested (Kotova, 2011).

H2Av (H2Az/H2Ax) may have other roles in the nucleus in addition to the regulation of PARP1. For instance, although the yeast genome does not encode any obvious PARP1 homolog, yeasts have H2Az (HTZ1) and H2Ax homologs (14). Moreover, both yeast histones play essential roles. Histone H2Ax phosphorylation is involved in genotoxic stress response, but HTZ1 regulates chromatin remodeling, transcription, and transcriptional silencing in heterochromatin. Although PARP1 is a target that performs a critical role in higher-order chromatin, which otherwise cannot be accomplished by yeast, these observations suggest that the function of H2Av may not be restricted to PARP1 activation (Kotova, 2011).

Because PARP1 activation has been shown in this work to be mediated through H2Av phosphorylation, it was further asked what signaling pathway and kinases might be responsible for such H2Av phosphorylation. During genotoxic stress response, cell-cycle checkpoint kinases such as ataxia telangiectasia-mutated/ataxia telangiectasia and Rad- related (ATM/ATR) and DNA-PK kinase, are shown to phosphorylate the C-terminal tail of H2Ax. Although the possible roles of the Drosophila homolog of these enzymes in chromatin regulation and transcription cannot be excluded, kinases such as Jil-1 kinase, which functions inside the puffs of polytene chromosomes, seem to be more promising candidates for performing this function. Therefore, one of the future directions for investigating the mechanism of PARP1 regulation in chromatin is to identify the kinase enzyme responsible for triggering H2Av-mediated PARP1 activation (Kotova, 2011).

The current paradigm for the role of the PARP1 protein has two parts. The first part assigns to PARP1 the role of DNA repair and genotoxic stress response, and the second part assigns to PARP1 functional roles in the regulation of chromatin structure and transcription. In demonstrating that phosphorylation of H2Av (the H2Az/H2Ax homolog) controls the activity of the PARP1 protein in both pathways, a more universal mechanism has been established for PARP1 regulation. These findings also support the notion that PARP1 is not simply a component of either the DNA-repair or transcriptional complexes but instead is a universal regulator of high-order chromatin, which in eukaryotes needs management during both DNA repair and transcription. The activation of the PARP1 protein by histone H2Av phosphorylation ultimately leads to the loosening of compacted chromatin and opens access for either the DNA repair machinery or the transcriptional apparatus (Kotova, 2011).

Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci

To efficiently transcribe genes, RNA Polymerase II (Pol II) must overcome barriers imposed by nucleosomes and higher-order chromatin structure. Many genes, including Drosophila Hsp70, undergo changes in chromatin structure upon activation. To characterize these changes, the nucleosome landscape of Hsp70 was mapped after an instantaneous heat shock at high spatial and temporal resolution. Surprisingly, an initial disruption of nucleosomes was found across the entire gene within 30 s after activation, faster than the rate of Pol II transcription, followed by a second further disruption within 2 min. This initial change occurs independently of Pol II transcription. Furthermore, the rapid loss of nucleosomes extends beyond Hsp70 and halts at the scs and scs' insulating elements. An RNAi screen of 28 transcription and chromatin-related factors reveals that depletion of heat shock factor, GAGA Factor, or Poly(ADP)-Ribose Polymerase or its activity abolishes the loss of nucleosomes upon Hsp70 activation (Petesch, 2009; full text of article).

Using a high resolution in vivo approach to map changes in the chromatin structure of the rapidly induced Hsp70 gene, a broad disruption of nucleosome structure was observed that occurred at a rate faster than transcribing Pol II and broader than a single transcription unit, ceasing at the natural insulating elements. Furthermore, it was found that the initial changes in chromatin architecture at Hsp70 can be decoupled from transcription of the gene, whereas the second disruption by 2 minutes is transcription-dependent. A selective RNAi screen identified HSF, GAF, and PARP as each being necessary for the changes in chromatin landscape at Hsp70 (Petesch, 2009).

Before HS, the Hsp70 gene contains a chromatin landscape that has many general, as well as some distinct features. Like many other TATA containing genes, a highly positioned nucleosome exists downstream of the promoter region and the adjacent nucleosomes on the body of Hsp70 gradually lose their positioning. Likewise, as seen with many genome-wide studies, the promoter, and a region at the 3' end of the gene, is relatively nucleosome free. It is yet to be determined why 3' ends of genes are hypersensitive to nucleases. However, while many genes in yeast contain a positioned nucleosome starting within the first 100 bp of the transcription unit, Hsp70 contains a nucleosome free region that extends further, with the first nucleosome centered 330 bp following the TSS. This extended nucleosome-free region may be a more general feature of genes containing a paused polymerase (Petesch, 2009).

The HS time course shows that within 2 minutes following HS, the chromatin landscape of Hsp70 drastically changes. Following 2 minutes of HS, there no longer exists appreciable protection of a contiguous 100 bp piece of DNA that would normally be provided from a histone octamer. However, there are still detectable levels of histone H3 on the body of the gene, albeit three-fold less than NHS levels. Although these results differ from early observations that histone levels on Hsp70 do not change following HS, the 3-fold decrease measured by qPCR agrees with more recent quantifications of histone levels following HS and may have gone undetected in the qualitative analysis of these early experiments. Early electron microscopy spreads of native transcribing Pol II complexes with a growing RNA chain from D. melanogaster indicate that the bulk of transcribing Pol II in vivo appears to have nucleosomes flanking its path. The current results, however, suggest that at least for the rapidly induced Hsp70 gene, the nucleosomal structure present before HS no longer exists following activation of the gene (Petesch, 2009).

Changes found in chromatin upon Hsp70 induction extend well beyond the transcription unit of Hsp70 and halt at the scs and scs' insulating elements. Previous studies of scs and scs' have shown that these insulators are capable of blocking enhancer functions and establishing chromatin domains that are resistant to position effects. However, the scs and scs' regions have been located by DNA FISH on squashed polytene chromosomes to be within a HS puff at the endogenous 87A HS locus. This indicates that the scs and scs' regions by themselves are not absolute boundaries to changes in chromosome architecture, and supports the observation that puffing is maximal at a time well after nucleosome disruption and therefore denotes additional structural alterations beyond those observed here. Although transcription of CG31211, CG3281, and Aurora did not change following HS, and no factor targeted for RNAi permitted the disruption of nucleosomes beyond scs or scs', both of these regions include a TSS with detectable amounts of Pol II. It is therefore possible that the promoter architecture with Pol II present at these genes may be responsible for establishing a barrier at these sites. Overall, the results show that scs and scs' provide a primary barrier to the spread of chromatin decondensation, at least at the nucleosomal level, and add to the limited knowledge of the chromatin architecture of a puff (Petesch, 2009).

The results indicate that transcription-independent chromatin decondensation may prove more general than previous believed. Changes in chromatin structure independent of transcription have been implicated at Hsp70 in humans and also at developmentally regulated puffs in Drosophila. Furthermore, the current results indicate that the changes in chromatin at D. melanogaster Hsp70 do not depend on many different transcription factors. In Saccharomyces cerevisiae, many HS genes also lose histone density within the body of the gene by 2 minutes of HS, and as in the current study, these changes are independent of SWI/SNF, Gcn5, and Paf1. Overall, transcription-independent chromatin decondensation might allow cells to rapidly activate genes by clearing the obstacles in the path of Pol II prior to its movement, together with its entourage of elongation factors, through the gene (Petesch, 2009).

The current results show that in addition to HSF and GAF, which have previously been implicated in the decondensation at Hsp70 loci, PARP is also necessary for rapid changes in the nucleosome architecture of Hsp70. This is consistent with the finding that reduction of PARP expression results in decreased HS puff sizes. The results go further in demonstrating that PARP aids the rapid removal of nucleosomes within 2 minutes of HS. Poly(ADP-)Ribose (PAR) polymers are the enzymatic product of PARP and have similar chemical and structural features as a nucleic acid. Upon activation, PARP polyribosylates itself, which results in PARP's release from chromatin. The result of this could be two fold. First, since PARP binds to nucleosomes in a similarly repressive manner as linker histone H1, the activation of PARP could result in its release from chromatin to reverse any repressive effects on the chromatin structure at Hsp70. Second, the ADP-ribosylation of histones may destabilize the nucleosome, and the creation of these PAR polymers could act locally as a nucleic acid that attracts and removes histones from the body of the Hsp70 gene. Alternatively, PARP could covalently modify another protein to activate its role in removal of nucleosomes (Petesch, 2009).

In addition to histones, PAR could also attract transcription factors that bind nucleic acids. This could explain the rapid recruitment of Pol II and other important transcription factors to the site of active HS transcription. Likewise, PAR could also provide a means through which transcription factors recruited to the gene are then retained locally. The activation of PARP could thus provide a rapid, transcription-independent method to deplete histones and promote transcription of the Hsp70 gene (Petesch, 2009).

Stress-induced PARP activation mediates recruitment of Drosophila Mi-2 to promote heat shock gene expression

Eukaryotic cells respond to genomic and environmental stresses, such as DNA damage and heat shock (HS), with the synthesis of poly-[ADP-ribose] (PAR) at specific chromatin regions, such as DNA breaks or HS genes, by PAR polymerases (PARP). Little is known about the role of this modification during cellular stress responses. This study shows that the nucleosome remodeler dMi-2 is recruited to active HS genes in a PARP-dependent manner. dMi-2 binds PAR suggesting that this physical interaction is important for recruitment. Indeed, a dMi-2 mutant unable to bind PAR does not localise to active HS loci in vivo. Several dMi-2 regions have been identified that bind PAR independently in vitro, including the chromodomains and regions near the N-terminus containing motifs rich in K and R residues. Moreover, upon HS gene activation, dMi-2 associates with nascent HS gene transcripts, and its catalytic activity is required for efficient transcription and co-transcriptional RNA processing. RNA and PAR compete for dMi-2 binding in vitro, suggesting a two step process for dMi-2 association with active HS genes: initial recruitment to the locus via PAR interaction, followed by binding to nascent RNA transcripts. It is suggested that stress-induced chromatin PARylation serves to rapidly attract factors that are required for an efficient and timely transcriptional response (Murawaska, 2011).

Mi-2 is strongly linked to transcriptional repression in both vertebrate and invertebrate organisms. Within NuRD and dMec complexes it contributes to the repression of cell type-specific genes. Therefore, the widespread colocalisation of dMi-2 with active Pol II and elongation factors at many chromosomal sites is surprising and suggests that dMi-2 might play an unappreciated role during active transcription, at least (or specifically) during environmental stresses such as HS. Indeed, dMi-2 is recruited to HS genes within minutes of HS. This property is not shared by other chromatin remodelers: Brahma (BRM) is not enriched at HS puffs and HS gene activation is independent of BRM function. Moreover, although imitation switch (ISWI) containing complexes are important for HS gene transcription, ISWI does not accumulate to high levels at active HS loci. Recruitment to HS puffs has previously been reported for Drosophila CHD1. Thus, accumulation at active HS genes is shared by at least two members of the CHD family of nucleosome remodelers but not by SWI/SNF and ISWI proteins (Murawaska, 2011).

Depletion of dMi-2 or a reduction of dMi-2 recruitment does not significantly perturb hsp70 transcription in Kc cells and, therefore, dMi-2 is dispensable for HS gene activation in this system. By contrast, depletion of dMi-2 in larvae strongly decreases hsp70, hsp26 and hsp83 activation. It is possible, that the RNAi-mediated depletion of dMi-2 is more efficient in transgenic flies compared to cell lines. In addition, it is believed that several factors contributing to HS gene activation are highly abundant or redundant in Kc cells but more limiting in other contexts. Accordingly, FACT and Spt6 are required for a HS gene activation in flies but are not essential in Kc cells (Murawaska, 2011).

The strong decrease of HS gene activation in dMi-2 RNAi larvae indicates a positive contribution of dMi-2 to transcription in vivo. Overexpression of inactive dMi-2 also results in reduced HS gene transcription implying that its enzymatic activity is critical. It is presently unclear whether this reflects a requirement for dMi-2 catalysed nucleosome remodeling or whether its activity is directed towards different substrates (Murawaska, 2011).

While dMi-2 could indirectly influence transcription by remodeling nucleosomes within the transcribed part of hsp70, its physical association with nascent HS gene transcripts argues for a more direct effect. Indeed, dMi-2 is not only required for high HS gene mRNA levels, but also affects the efficiency of co-transcriptional 3' end formation and splicing. A role of chromatin remodelers in splicing has been suggested before: Both CHD1 and BRG1 bind components of the splicing apparatus. CHD1 associates with Pol II and binds nucleosomes containing H3K4me3, which are enriched near the 5' end of active genes . BRG1 is present at the coding region of genes and influences splice site choice. It has been proposed that CHD1 and BRG1 physically recruit splicing factors but it is unclear if their ATPase activities play a role. Indeed, inactive BRG1 retains the ability to affect exon choice. Inefficient processing of the hsp70 and hsp83 transcripts is not only observed in larvae expressing reduced levels of dMi-2. Importantly, even stronger processing defects are generated by overexpression of inactive dMi-2. This strongly suggests, for the first time, that the catalytic activity of a chromatin remodeler is required for correct co-transcriptional RNA processing. It remains to be determined whether dMi-2 nucleosome remodeling activity influences RNA processing indirectly, e.g. by altering Pol II elongation rates, or whether it has a more direct role (Murawaska, 2011).

A series of complementary results support the hypothesis that dMi-2 interacts with PAR polymers that are rapidly synthesized at activated HS loci. First, the broad distribution of dMi-2 over the entire transcribed region correlates with the distribution of PAR polymer. Second, pharmacological inhibition of PARP greatly decreases dMi-2 binding to activated hsp70. Third, dMi-2 directly binds PAR polymers in vitro. Fourth, a dMi-2 mutant unable to bind PAR also fails to localise to active HS loci. dMi-2 physically associates with nascent HS gene transcripts and binds RNA in vitro. While this interaction is potentially important for the efficiency of transcription and processing, it likely plays a minor role in dMi-2 targeting. Accordingly, inhibition of transcriptional elongation has no significant effect on dMi-2 recruitment (Murawaska, 2011).

It is important to note, that while the results argue for an important role of PAR binding in the recruitment of dMi-2 to HS loci, it cannot be excluded that protein-protein interactions with histone or non-histone proteins also play a role (Murawaska, 2011).

This analysis indicates that dMi-2 harbours several PAR binding motifs in its N-terminal region. It has been demonstrated that human CHD4 is recruited to double stranded DNA breaks in a PARP-dependent manner (Polo, 2010). That study demonstrated PAR binding activity to the region N-terminal of the ATPase domain of CHD4. This agrees well with the current data and suggests that the PAR binding function of CHD4/dMi-2 has been conserved in evolution (Murawaska, 2011).

Two structural protein modules directly interact with PAR, the macrodomain and the PBZ domain; however, these domains are not present in dMi-2. In addition, several shorter PAR binding motifs have been identified. These motifs bear little sequence similarity but share the presence of several K/R residues which are interspersed by hydrophobic residues. The current results have uncovered three K/R-rich regions with PAR binding activity near the N-terminus of dMi-2. Two of these three K/R-rich regions (K/R III and K/R IV) consist of interspersed basic and hydrophobic residues and are therefore reminiscent of the previously described PAR binding motifs, and the third (K/R I) lacks hydrophobic residues completely. None of the three K/R regions matches the consensus PAR binding motifs. It is possible that a consensus motif should generally be chosen less stringently and that a high content of K and R-residues in these regions is sufficient to provide PAR binding activity in vitro. Further characterisation of these regions will be required to resolve this issue. In addition to the K/R regions, the tandem chromodomains of dMi-2 bind PAR in vitro. Previous studies have shown that the chromodomains are required for interacting with nucleosomal DNA in vitro. The new data suggests that these domains can interact with different nucleic acids (Murawaska, 2011).

Several potential molecular functions of PARylation at HS genes have been suggested. First, PARP activity is required for the rapid loss of nucleosomes at hsp70 within the first two minutes after HS (Petesch, 2008). It has been suggested that PARylation of histones aids rapid nucleosome disassembly (Petesch, 2008). Second, at later stages of the HS response (20-60 minutes after HS), PARP activity is required to establish a compartment which restricts the diffusion of factors such as Pol II and Spt6 and promotes efficient factor recycling. The current results suggest that PARylation carries out a third task, namely, to recruit factors via their direct interaction with PAR. The earliest time point when dMi-2 binding to hsp70 can be detected is between 2 and 5 minutes after HS. This places dMi-2 recruitment between the early PARP-dependent nucleosome removal (0-2 minutes after HS) and effects of the transcription compartment (20-60 minutes after HS) (Murawaska, 2011).

The ability of dMi-2 to bind both PAR and RNA and the finding that RNA can compete for PAR binding to dMi-2 is consistent with the hypothesis that dMi-2 association with active HS genes is a two step process. It is proposed that dMi-2 is initially recruited via interaction with PAR polymers. Synthesis of these starts prior to the onset of hsp70 transcription (Petesch, 2008). This results in a rapid local increase of the dMi-2 concentration. In the second step, when hsp70 transcripts are produced by elongating RNA polymerase II at high rates, dMi-2 can switch from binding PAR to interacting with nascent transcripts (Murawaska, 2011).

Severe cellular stresses, such as DNA strand breaks and acute HS, must be dealt with quickly and efficiently. In both cases, a multitude of factors are rapidly recruited to orchestrate the repair of DNA and the massive transcriptional activation of HS genes, respectively. It is postulated that rapid synthesis of PAR polymers at both DNA damage sites and HS genes affords an efficient mechanism to recruit chromatin remodelers and other factors. It has recently been shown that PARylation of DNA breaks is instrumental in recruiting chromatin remodelers, including mammalian dMi-2 homologs, to damaged sites. This study shows that dMi-2?s recruitment to activated HS genes requires PARP activity and that dMi-2 binds PAR directly. The high local concentration of PAR polymers at DNA breaks and HS genes might exploit the general affinity of dMi-2 for nucleic acids. Indeed, dMi-2 binds both DNA and RNA as well as PAR in vitro. In this manner, PAR polymers might act as a scaffold to redirect dMi-2 to chromatin regions where high levels of dMi-2 activity are required, thus acting as a stress-dependent, transient affinity site for chromatin remodeling and possibly RNA processing activities. The results highlight a signaling and scaffolding function for PARP activity during transient environmental stresses other than DNA damage, suggesting that PARylation carries out important modulatory functions in the stress-dependent reprogramming of nuclear activities (Murawaska, 2011).

Functional analysis of Drosophila BRCA2 in DNA repair

The human BRCA2 cancer susceptibility protein functions in double-strand DNA break repair by homologous recombination and this pathway is conserved in the fly Drosophila. Although a potential Drosophila BRCA2 orthologue (Brca2; CG30169) has been identified by sequence similarity, no functional data addressing the role of this protein in DNA repair is available. This study demonstrates that depletion of Brca2 from Drosophila cells induces sensitivity to DNA damage induced by irradiation or treatment with hydroxyurea. Brca2 physically interacts with rad51 (spnA), and the two proteins become recruited to nuclear foci after DNA damage. A functional assay for DNA repair demonstrated that in flies Brca2 plays a role in double-strand break repair by gene conversion. Finally, it was shown that depletion of Brca2 in cells is synthetically lethal with deficiency in other DNA repair proteins including parp. The conservation of the function of BRCA2 in Drosophila will allow the analysis of this key DNA repair protein in a genetically tractable organism potentially illuminating mechanisms of carcinogenesis and aiding the development of therapeutic agents (Brough, 2008).

The Drosophila genome carries a potential BRCA2 orthologue as indicated by the presence of BRC (RAD51-binding) motif sequences (Lo, 2003). However, this protein does not contain recognisable DNA and DSS1 binding domains, both characteristics of the mammalian BRCA2 protein. Using both cell culture and whole organism genetic approaches this study has shown that despite lacking these motifs the CG30169 allele is the likely functional BRCA2 orthologue. Using Drosophila cells in culture it was shown that a deficiency for the Brca2 protein induces sensitivity to both X-rays and the DNA-damaging drug HU. This phenotype is typical of eukaryotic cells deficient in DNA repair and has been demonstrated using various DNA damaging agents in the fly for a number of mutant genes, including rad51. By comparison, only a few studies have shown a heightened sensitivity to DNA damage in fly cells in culture (Brough, 2008).

I-SceI-based assays were subsequently performed to investigate the role of Drosophila BRCA2 in DSBR. The results clearly showed that Brca2 is essential at least for inter-homolog gene conversion repair. In this respect, the Brca2 mutant behaves similarly to Drosophila rad51 and rad54 mutations. These results are also consistent with those from mammalian and fungal studies. Therefore, it is concluded that the essential function of Brca2 in homology-directed DSBR is evolutionarily conserved despite poor conservation in protein sequence (Brough, 2008).

Further evidence that Brca2 is involved in DNA repair was provided by investigating a possible interaction between Brca2 and Rad51. Co-immunoprecipitation showed that the two proteins interact in both the presence and absence of DNA damage. However, using immunofluorescence analysis it was demonstrated that the proteins co-localise within nuclear foci following DNA damage but not before and that Brca2 is likely to be involved in the recruitment of Rad51 to the sites of damage. The interaction of Brca2 and Rad51 is consistent with the presence of three BRC repeats within Brca2. However, Brca2 unlike other BRCA2 orthologues lacks a recognisable OB fold domain capable of binding DNA. It seems possible Brca2 interacts with another protein which performs this function (Brough, 2008).

Heterozygous germline mutations of the BRCA2 gene in humans confer a high risk to a range of cancers. The mechanism for this is through genome instability caused by loss of the wild-type BRCA2 allele in tumours. One approach to the development of new therapeutic approaches is to target the deficiency in DNA repair. Such synthetic lethal therapeutics are in development via the inhibition of the enzyme PARP which is involved in base excision repair. To extend this approach it is important to identify additional synthetic lethal interactions. Drosophila cells have already been used to identify evolutionarily conserved pathways and genetic interactions. Therefore, to test the feasibility of such an approach in DNA repair pathways the synthetic lethal interaction of Brca2 deficiency with a number of DNA repair genes was studied (Brough, 2008).

This study shows that synthetic lethal interactions exist between Brca2 and Parp, analogous to the mammalian system. This suggests that the interactions between DNA repair pathways are evolutionarily conserved. In addition, an interaction between alternative dsDNA break repair pathways (NHEJ) was observed. Similar synergy between the HR and NHEJ pathway has already been observed in Drosophila; for instance, crossing Blm or LigIV mutant flies with Rad54 mutant flies was shown to increase the sensitivity of the resulting progeny to DNA damage. The functional conservation of BRCA2 as well as the conserved interplay of HR with other DNA repair pathways, as demonstrated by synthetic lethal interactions, suggests that Drosophila will be a powerful system for dissecting BRCA2 biology as well as aiding the development of new therapeutic approaches (Brough, 2008).

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).

NAD+-dependent modulation of chromatin structure and transcription by nucleosome binding properties of PARP-1

PARP-1 is the most abundantly expressed member of a family of proteins that catalyze the transfer of ADP-ribose units from NAD+ to target proteins. This study describes previously uncharacterized nucleosome binding properties of PARP-1 that promote the formation of compact, transcriptionally repressed chromatin structures. PARP-1 binds in a specific manner to nucleosomes and modulates chromatin structure through NAD+-dependent automodification, without modifying core histones or promoting the disassembly of nucleosomes. The automodification activity of PARP-1 is potently stimulated by nucleosomes, causing the release of PARP-1 from chromatin. The NAD+-dependent activities of PARP-1 are reversed by PARG, a poly(ADP-ribose) glycohydrolase, and are inhibited by ATP. In vivo, PARP-1 incorporation is associated with transcriptionally repressed chromatin domains that are spatially distinct from both histone H1-repressed domains and actively transcribed regions. Thus, PARP-1 functions both as a structural component of chromatin and a modulator of chromatin structure through its intrinsic enzymatic activity (Kim, 2004).

Search PubMed for articles about Drosophila Parp

Brough, R., Wei, D., Leulier, S., Lord, C. J., Rong, Y. S. and Ashworth, A. (2008). Functional analysis of Drosophila melanogaster BRCA2 in DNA repair. DNA Repair (Amst) 7: 10–19. PubMed Citation: 17822964

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

Kotova, E., et al. (2011). Drosophila histone H2A variant (H2Av) controls poly(ADP-ribose) polymerase 1 (PARP1) activation in chromatin. Proc. Natl. Acad. Sci. 108(15): 6205-10. PubMed Citation: 21444826

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

Petesch, S. J. and Lis, J. T. (2008). Rapid, transcription-independent loss of nucleosomes over a large chromatin domain at Hsp70 loci. Cell 134: 74-84. PubMed Citation: 18614012

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

Polo, S. E., Kaidi, A., Baskcomb, L., Galanty, Y. and Jackson, S. P. (2010). Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4. Embo J. 29: 3130-3139. PubMed Citation: 20693977

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

Zhang, J. (2003). Are poly(ADP-ribosylation) by PARP-1 and deacetylation by Sir2 linked? BioEssays 25: 808-814. PubMed Citation: 12879452

Ziegler, M. and Oei, S. L. (2001). A cellular survival switch: poly(ADP-ribosyl)ation stimulates DNA repair and silences transcription. BioEssays 23: 543-548. PubMed Citation: 11385634

date revised: 10 February 2012

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