Review The taming of PARP1 and its impact on NADþ metabolism Sarah Hurtado-Bagès 1, 2, 7, Gunnar Knobloch 3, 8, Andreas G. Ladurner 3, 4, 5, **, Marcus Buschbeck 1, 6, * ABSTRACT Background: Poly-ADP-ribose polymerases (PARPs) are key mediators of cellular stress response. They are intimately linked to cellular metabolism through the consumption of NADþ. PARP1/ARTD1 in the nucleus is the major NADþ consuming activity and plays a key role in maintaining genomic integrity. Scope of review: In this review, we discuss how different organelles are linked through NADþ metabolism and how PARP1 activation in the nucleus can impact the function of distant organelles. We discuss how differentiated cells tame PARP1 function by upregulating an endogenous inhibitor, the histone variant macroH2A1.1. Major conclusions: The presence of macroH2A1.1, particularly in differentiated cells, raises the threshold for the activation of PARP1 with consequences for DNA repair, gene transcription, and NADþ homeostasis. Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords PARP1; NADþ; Epigenetics; MacroH2A; Metabolism; Homeostasis 1. PARP1 IS A KEY MEDIATOR OF NUCLEAR POLY-ADP- 1.1. The family of PARP enzymes RIBOSE SIGNALING The PARP family of ARTs, also known as ARTD [12], catalyzes the modification of proteins through the addition of one or more ADP- The covalent linkage of adenosine diphosphate (ADP)-ribose units to ribose groups (Figure 1). In humans, 17 members of this family of effector molecules such as proteins, nucleic acids, or small molecules enzymes have been identified and are classified according to their is a reversible, evolutionarily conserved chemical modification found in activity [13]: PARP1, 2, 5a, and 5b catalyze the polymerization of ADP- all kingdoms of life (reviewed in [1]). The ADP-ribosylation of proteins, ribose units (PARylation) into linear or branched chains through a(1e2) a post-translational modification, has been implicated in many phys- O-glycosidic bonds of neighboring ribose moieties. All other PARPs iological processes, including gene transcription, protein degradation, with the exception of PARP9 and PARP13 catalyze the addition of single cell proliferation and differentiation, DNA damage and repair, aging, ADP-ribose units onto target proteins through a process that is inflammation, cell death, hostevirus interactions, and metabolism [2e generally referred to as mono-ADP-ribosylation or MARylation. To date, 6]. The reaction is catalyzed by ADP-ribosyltransferases (ARTs) that no enzymatic activity has been detected for PARP9 and PARP13 [7]. In include most members of the poly-ADP-ribose polymerase (PARP) addition to their conserved catalytic ART domain, PARPs contain family of proteins as well as some members of the sirtuin family [7,8]. additional structural domains that confer distinct localizations and Although PARPs and sirtuins differ substantially in their protein functions or tightly regulate their spatiotemporal activity [14]. Based on structure, they both use NADþ as a substrate (Figure 1). Unlike PARPs, their domain architecture, PARPs can be sub-divided into four groups: the majority of sirtuins use NADþ for deacetylation and not ADP- DNA-dependent PARPs 1e3, ankyrin repeat containing tankyrases ribosylation. This study focuses on PARPs. The function of sirtuins PARP5a and b, Cys-Cys-Cys-His zinc finger containing PARP7, and their crosstalk with PARPs was previously reviewed elsewhere PARP12, and PARP13, and macrodomain containing PARPs 9, 14, and [9e11]. 15. The most abundant and best-characterized family member is 1 Josep Carreras Leukemia Research Institute, Campus ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916, Badalona, Spain 2Department of Experimental and Health Sciences, Universitat Pompeu Fabra (UPF), Barcelona, Spain 3Biomedical Center Munich, Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany 4Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität München, 81377, Munich, Germany 5Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-Universität München, 81377, Munich, Germany 6Program for Predictive and Personalized Medicine of Cancer, Germans Trias i Pujol Research Institute (PMPPC-IGTP), 08916, Badalona, Spain 7 Current address: sci.sters.editions Company, Graphic Design and Illustration for Science, 42100, Saint-Étienne, France. 8 Current address: Eisbach Bio GmbH, Am Klopferspitz 19, 82152, Planegg-Martinsried, Germany. *Corresponding author. Josep Carreras Leukemia Research Institute, Campus ICO-Germans Trias i Pujol, Universitat Autònoma de Barcelona, 08916, Badalona, Spain. E-mail: mbuschbeck@carrerasresearch.org (M. Buschbeck). **Corresponding author. Biomedical Center Munich, Physiological Chemistry, Ludwig-Maximilians-Universität München, 82152, Planegg-Martinsried, Germany. E-mail: andreas.ladurner@bmc.med.lmu.de (A.G. Ladurner). Received October 18, 2019  Revision received January 10, 2020  Accepted January 24, 2020  Available online 12 February 2020 https://doi.org/10.1016/j.molmet.2020.01.014 MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1 www.molecularmetabolism.com Review Figure 1: PARPs catalyze poly-ADP-ribosylation. Several members of the PARP family are able to generate linear and branched ADP-ribose chains. PARP1 provides the major PARP activity in the nucleus. It transfers the ADP-ribose moiety of NADþ onto substrates that can be other ADP-ribose units (poly-ADP-ribosylation) or proteins. Nicotinamide is the side product of this reaction. PARP1, a nuclear enzyme with diverse cellular functions ranging from ribose moieties in length [19]. This creates an interaction scaffold for DNA repair, transcriptional control, genome stability, and cell death PAR binding DNA repair proteins that are recruited to the sites of (reviewed in [15]). PARP1 activity. These include important DNA repair factors such as XRCC1, APLF, CHFR, MRE11, and ATM that can be attracted to PAR 1.2. Activation of PARP1 by DNA damage synthesis sites through their linear, low-complexity PAR binding motifs The PARP1 protein consists of multiple functional domains: an N-ter- (summarized in [20]). It has also been proposed that high local PAR minal DNA binding domain composed of three zinc finger motifs (Zn1- concentrations can induce a compartmentalization at the DNA break 3), an auto-modification domain, and the C-terminal ART domain. The through liquideliquid phase separation, attracting positively charged catalytic ART domain is further composed of a conserved tryptophan, intrinsically disordered proteins such as the FET family of RNA binding arginine, and glycine-rich WRG domain and a regulatory helical domain proteins, which are required for DNA repair [21]. “Reader” proteins (HD). The HD inhibits substrate binding to the catalytic ART domain containing macrodomains, PAR binding zinc finger (PBZ) domains, or [16]. The zinc fingers Zn1 and Zn2 are sequence-independent sensors tryptophan containing WWE domains specifically recognize mono- or of DNA strand breaks that recruit PARP1 to a variety of DNA damage poly-ADP-ribosylation through the direct engagement of the PAR structures, including double strand breaks (DSB) and nicked or gapped polymer in their binding pockets (reviewed in [22]). In addition to the single strand breaks (reviewed in [17]). The binding of Zn1 and Zn2 to highly specific interaction of PAR reader modules with PAR nucleic DNA creates a domainedomain interaction surface that directs the acid, the high density of negative charges in the PAR chains leads to structural rearrangement of the remaining enzyme into its activated chromatin expansion and can dissociate DNA from histone octamers conformation, ultimately resulting in a destabilization of the auto- and release PARP1 from DNA damage sites, which downregulates the inhibitory HD domain and allowing NADþ binding to the catalytic site activity of the PARP1 enzyme [23]. Several studies clearly showed that [18]. The fully activated PARP1 subsequently modifies itself and target the compact chromatin structure is additionally relaxed by the proteins with long, branched PAR chains that can span up to 200 ADP- recruitment of specific chromatin remodeling enzymes such as ALC1/ 2 MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com CHD1L or CHD2 that promote chromatin expansion [24,25] and in- 2.1. Regeneration of the NADþ pool crease accessibility of DNA for repair proteins [26,27]. PAR-activated Steady-state cellular NADþ levels are between 200 and 500 mM [44]. signaling is finally terminated by the PAR hydrolyzing enzymes PARG Most cells replenish their NADþ pool primarily through a salvage and ARH3 [28,29]. These so-called “erasers” create locally high pathway and to a lesser extent through de novo synthesis from dietary concentrations of ADP-ribose that can be converted into ATP by the sources. Dietary sources are nicotinamide (NAM) and nicotinic acid and pyrophosphatase NUDIX5 [30]. This nuclear ATP source has been are commonly referred to as vitamin B3 and their ribosides and the proposed to fuel the DNA repair machinery, chromatin remodeling, and amino acid tryptophan [45]. The NADþ salvage pathway allows the hormone-induced transcriptional regulation. recycling of NAM that is the product generated by NADþ consuming enzymes. The rate-limiting step is the conversion of NAM into nico- 1.3. Regulation of PARP1 and its role in transcription tinamide mononucleotide (NMN) by nicotinamide phosphoribosyl- PARP1 is an important mediator of extra- and intracellular stress transferase (NAMPT) [46]. Three nicotinamide mononucleotide signals. The cellular outcome depends on the level of PARP1 activation adenylyltransferases (NMNAT1-3) catalyze the conversion of NMN to and can range from activation of the DNA damage repair machinery to NADþ. The recycling of one molecule of NADþ from NAM comes at the initiation of cell death [31,32]. In addition to the allosteric activation by cost of two ATP molecules. DNA breaks, PARP1 activity is subjected to regulatory mechanisms through post-translational modifications and proteineprotein in- 2.2. Compartmentalization of NADþ metabolism and synthesis teractions. Methylation by Set7/9 [33], phosphorylation by ERK1/2 [34], Most metabolic pathways involving NADþ/NADH-dependent redox or its interaction with phosphorylated ERK2 [35] increase PARP1 ac- reactions occur in mitochondria and cytosol [45]. In addition to mito- tivity. In contrast, the interaction of PARP1 with HPF1 promotes the chondria and cytoplasm, a substantial amount of NADþ is consumed in PARylation of histones, reduces its auto-modification activity, and the nucleus. Nuclear NADþ consuming enzymes include PARP1, generally shifts the specificity of PARP1 from glutamate, aspartate, and PARP2, and the sirtuins SIRT1 and SIRT2. Hence, the regeneration of lysine residues to serines as main PAR acceptor sites [36,37]. As NADþ through the salvage pathway is compartmentalized (Figure 2). further discussed in Section 4, histone variant macroH2A1.1 dampens While the NAMPT enzyme is located in the cytosol and nucleus, the PARP1 activity by binding its auto-modified form [38,39]. three NMNAT enzymes have a distinctly subcellular distribution. An additional important function of PARP1 is its role as a transcriptional NMNAT1 is nuclear, NMNAT2 is located in the cytoplasm and at the regulator. Two general mechanisms have been proposed for the external membrane of the Golgi, and NMNAT3 localizes inside mito- function of PARP1: (1) by regulation of the local and higher order chondria [47]. Interestingly, PARP1 has been shown to directly interact chromatin structure through direct interaction with nucleosomes or with NMNAT1 and recruit it to specific promoter sites where the locally PARylation of histones and chromatin-associated factors, and (2) as a synthesized NADþ “feeds” the PARP1 catalysis-dependent transcrip- co-regulator through interactions with the transcriptional machinery or tional modulation of target genes [48]. Importantly, the NMNATs of all sequence specific transcription factors [6,40]. While most studies cell compartments compete for the same pool of NMN produced by show a requirement of PARP1 catalytic activity for its transcriptional NAMPT thereby placing the intermediate in a central position of linking function [41,42], others reported transcriptional modulation indepen- NADþ metabolism of different compartments. This is illustrated by the dent of PARP activity [43]. observation that glucose-induced upregulation of cytosolic NMNAT2 in In sum, PARP1 is an abundant nuclear enzyme whose stress-induced adipogenic cells leads to reduced availability of NADþ in the nucleus activation during transcription or DNA damage leads to dramatic al- with consequences for PARP1-mediated gene regulation and cell dif- terations in nuclear structure and function and is closely intertwined ferentiation [49]. with cellular metabolism through the consumption of NADþ. In conclusion, reactions catalyzed by PARPs, sirtuins and cyclic ADP- ribose synthetases consume NADþ while redox reactions do not 2. NADþ METABOLISM change the overall pool of the NADþ/NADH pair. Separate NADþ pools exist in compartments and organelles. They are dynamic and Nicotinamide adenine dinucleotide (NADþ) is an essential cofactor communicate through common precursors of the salvage pathway. involved in most reductive-oxidative metabolic pathways including glycolysis and the citric acid cycle (reviewed in [44]). NADþ and its 3. THE IMPACT OF ACUTE PARP1 ACTIVATION ON reduced form NADH function as acceptor and donor molecules of two METABOLISM electrons and one proton. In the mitochondria, this function allows the coupling of the citric acid cycle to the electron transport chain, which is More than 50 years ago, NMN was shown to induce a nucleotide essential for the efficient conversion of nutrients into energy. The polymer when added to liver nuclear extract [50]. This nucleotide membrane potential generated by the electron transport chain drives polymer was later shown to be PAR. This was the first indication that oxidative phosphorylation generating adenosine triphosphate (ATP). the NADþ salvage pathway, PARP activity, and the generation of PAR In addition to its function in redox reactions, NADþ serves as an ADP- were linked to each other. In eukaryotic cells, PARP1 accounts for the ribose donor for enzymes regulating important biological processes largest amount of PARP activity in cells and is the major NADþ such as transcriptional regulation, calcium signaling, and DNA repair consuming entity in the nucleus [51]. Acute activation of PARP1 (reviewed in [45]). These enzymes include PARPs, the deacetylase through hormone stimulation or DNA damage can lead to the depletion family of sirtuins, and synthetases generating the second messenger of 50%e80% of total cellular NADþ [52,53]. PARP1 activation is cyclic ADP-ribose. In this section, we briefly discuss the synthesis of usually transient and NADþ levels are quickly recovered, within 4e5 h NADþ and the relevance of its subcellular compartmentalization for in cases of hormone stimulation [52]. However, prolonged activation of inter-organelle communication (Figure 2). Those interested in the PARP1 in cases of persistent stress can impair the energetic balance of nutritional aspects of NADþ and the possibilities for pharmacological cells [44], leading to severe ATP depletion and triggering apoptosis intervention should refer to two previous reviews [44,45]. [54]. MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 3 www.molecularmetabolism.com Review Figure 2: NADD metabolism connects cellular sub-compartments. Most cellular NADþ is regenerated through the salvage pathway from NAM and to a smaller extent through de novo biosynthesis from dietary sources such as NR (nutrient). The rate-limiting reaction from NAM to NMN is catalyzed by NAMPT enzyme in the nucleocytosol. Three different NMNAT enzymes catalyze the compartmentalized regeneration of NADþ from the same pool of NMN. The NADþ/NADH pair has key redox functions in the tricarboxylic acid (TCA) cycle and the electron transport chain to drive ATP generation. Conversely, inhibition of PARP1 can increase energy balance and Treatment of cell cultures with PARP inhibitors increased the improve mitochondrial function. We recently summarized the current cellular NADþ levels [52,59,61]. Interestingly, increased NADþ knowledge about PARP1 loss-of-function studies in mice [55]. availability frequently leads to higher mitochondrial activity. The Although the outcomes were variable and strongly diet and genetic Auwerx Lab showed an indirect effect on respiration exerted by context dependent, most of these studies suggested that PARP inhi- increased NADþ-dependent SIRT1 activity in the nucleus that is bition is metabolically favorable. Pharmacologic PARP inhibitors or mediated by the upregulation of genes promoting respiration genetic inhibition of PARP1 protect mice from diabetes when induced [59,62]. Studying differentiated muscle cells, we further found a by administration of the b-cell toxin streptozotocin [56,57]. Similarly, direct effect of inhibited PARP1 mediated by increased availability both PARP inhibitors and PARP1 knock-out reduce disease parameters of the NADþ precursor NMN in the cytosol and increased regen- in obesity-challenged mice. This includes reduced diabetes-associated eration of NADþ in the mitochondria [39,55]. kidney damage in the genetic Lbdr (db/db) model [58] and protection Taken together, PARP1 activity in the nucleus can affect the meta- against obesity in mice on high-fat diets [59,60]. bolism of distant compartments, notably mitochondrial respiration, In several cases, the favorable effect of PARP1 inhibition was through the consumption of NADþ and the depletion of precursors from linked to an increased availability of NADþ for other reactions. common pools. The inhibition of PARP1 is emerging as a potential 4 MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com therapeutic strategy to modulate NADþ levels and shift NADþ usage to macroH2A1.1 containing nucleosomes show an inhibition of the other reactions and compartments. associated PARP1 activity [38]. Using in vitro assays, we found that the macrodomain of macroH2A1.1 is sufficient for inhibiting PARP1 [39]. In 4. TAMING OF THE PARP1 STRESS RESPONSE BY THE both cases, the inhibitory capacity is strictly dependent on the integrity HISTONE VARIANT MACROH2A1.1 IN DIFFERENTIATED CELLS of the binding pocket and its capacity to bind ADP-ribose [38,39]. As such, the PARP inhibitory function is strictly limited to the splice iso- Terminally differentiated and proliferating cells have different re- form macroH2A1.1 (Figure 3). We and others were unable to confirm quirements for DNA repair and thus for DNA damage-induced PARP1 an earlier report suggesting that the macrodomains of the other signaling. While proliferating cells require fast repair until the next macroH2A proteins would be able to inhibit PARP1 [75]. entry into the S-phase, this is much less critical in non-cycling cells At present, it is unclear how exactly auto-modification and mac- that most likely acquire less DNA damage and might be able to tolerate roH2A1.1 binding modulate the activity of PARP1. But as increased it for a prolonged time. As such, in retrospect, it is not surprising that auto-modification correlates with increased activation, it is possible metazoan species have evolved an inhibitory mechanism dampening that auto-modification stabilizes the enzyme in its active conformation. but not abrogating PARP1 signaling in terminally differentiated states. We can further envision two possible scenarios for how the binding of In the context of chromatin, one of these endogenous inhibitors is the macroH2A1.1 ADP-ribosylated to PARP1 interferes with its activity: (i) histone variant macroH2A1.1. The close binding of a single macrodomain to mono-ADP-ribosylated PARP1 or a form of PARP1 modified with a short PAR chain directly 4.1. MacroH2A histone variants affects the conformation and activity of the enzyme. (ii) The binding of The exchange of a replication-coupled histone for a histone variant is the macrodomains to all ends of growing PAR chains deprives PARP1 one of the most drastic changes that can occur on the level of the of access to its target site, which does not allow sufficient auto- nucleosome and endow the immediate chromatin environment with modification to stabilize the active state. A major difference between new structural and biophysical properties [63]. In particular, for H2A these two hypothetical mechanisms is that one-to-one binding would and H3, many histone variants exist. Among these, three macroH2A suffice for the first mechanism while the second would require an histone variants stick out because they have a unique tripartite excess of macroH2A1.1 macrodomains. Our observation that close to structure consisting of a histone-fold domain followed by a C-terminal equimolar levels of macroH2A1.1 were sufficient to inhibit PARP1 in unstructured linker connecting with a globular macrodomain [64]. The myotubes argues for the first and more direct mechanism [39] linker protrudes from the compact structure of the nucleosome near (Figure 3). In cases where macroH2A1.1 is present at lower levels than the dyad axis and places the macrodomain at an accessible position PARP1 or in situations where the PARP1-activating stimulus is too outside the chromatin fiber. MacroH2A proteins contribute to epige- strong, endogenous inhibition is not expected to occur. However, netic regulation in development, differentiation, and somatic cell macroH2A1.1 would still be able to recruit active and auto-modified reprogramming and cancer. Those interested in these aspects of forms of PARP1 with longer, multiple, and branched PAR chains to macroH2As should refer to two recent reviews in which we provided a genomic loci (Figure 3). This has the potential to explain how mac- synthesis of the current knowledge [63,65]. As a resource to the field, roH2A1.1 cooperates with PARP1 in gene regulation [38,42] as well as we published an overview of loss-of-function studies conducted in in DNA repair. Two examples of the latter are the promotion of cell mice highlighting differences in genetic context and experimental survival in response to ionizing radiation [76] and the repair of oxidative approaches [55]. The molecular mechanism by which macroH2As damage [77]. Taking all these studies together, the relative ratio of mediate their epigenetic function is yet unknown but is likely related to macroH2A1.1 and PARP1 decides whether macroH2A1.1 acts as in- a major role in regulating higher order chromatin structures including hibitor or cooperative partner of PARP1. Inhibition is limited to situa- heterochromatin [66e68]. tions in which the level of macroH2A1.1 is equal or exceeds the level of PARP1. 4.2. MacroH2A1.1 macrodomain binds and inhibits ADP- ribosylated PARP1 4.3. MacroH2A1.1 couples differentiated state and NADþ Two genes and one alternative splicing event produce three macroH2A metabolism proteins that differ in their macrodomains [69]. Macrodomains are We studied the PARP inhibitory function of macroH2A1.1 during the ancient globular folds that have a binding pocket for ADP-ribose [70]. terminal differentiation of myoblasts to myotubes. Our study suggested The capacity to bind ADP-ribose is limited to the splice variant mac- that this endogenous inhibition operates in terminally differentiated roH2A1.1 [68,71]. MacroH2A1.1 differs in only 26 amino acids from myotubes but not in proliferating myoblasts [39]. We found that the alternative splice variant macroH2A1.2 [72]. These amino acids macroH2A1.1 is barely expressed in fast proliferating cells [78] but form part of the binding pocket required for ADP-ribose binding [71]. rapidly upregulated through a switch in alternative splicing during The expression of both variants is tissue specific [72] and mac- myogenic differentiation, reaching levels matching those of PARP1 roH2A1.1 is highly expressed in terminally differentiated cells such as [39]. Inhibition of PARP1 by macroH2A1.1 leads to decreased NADþ enterocytes and myotubes [39,73]. The binding mode of ADP-ribose consumption in the nucleus and sustained levels of the precursor NMN. allows macroH2A1.1 to also bind the terminal part of PAR chains This in turn leads to increased pools of mitochondrial NADþ and and proteins post-translationally modified by ADP-ribosylation [74]. improved oxidative phosphorylation. The upregulation of macroH2A1.1 The latter has been shown for auto-modified PARP1. Although this has allows differentiated muscle cells to avoid unnecessary NADþ con- not been formally tested, the ADP-ribose bound structure of the sumption in the nucleus, prioritizing its use in the mitochondria for ATP macrodomain suggests that it would be compatible with binding production and cell survival. Several lines of observation suggest that mono-ADP-ribosylated proteins. All macroH2As co-purify with PARP1 this mechanism is general and not limited to muscle tissue. First, in the context of purified mono-nucleosomes; however, only macroH2A1.1 is also upregulated during differentiation of other cell MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 5 www.molecularmetabolism.com Review Figure 3: MacroH2A1.1 is an endogenous inhibitor of PARP1. NMNAT1 acts as feeder providing NADþ to PARP1. Activation of PARP1 is accompanied by auto-modification. Binding of the “tamer” macroH2A1.1 leads to its inhibition. The taming can be overcome by PARP1 hyperactivation and hypermodification. Proteins containing poly-ADP-ribose (PAR) interaction domains including macrodomains can act as readers of the ADP-ribose units. PARG and ARH3 are the erasers that remove PAR chains. types including embryonic stem cells, colon cancer cells, and adipo- nucleus is responsible for all reactions using genomic DNA as a cytes [73,78,79]. Second, the overexpression of macroH2A1.1 but not template, mitochondria are the cells’ major energy source. As such, it macroH2A1.2 in liver cells prevented the accumulation of fat most is not surprising that cells have evolved mechanisms to coordinate the likely by promoting its oxidation in mitochondria [80,81]. activities of both organelles. Although mitochondria are a clearly Taken together, differentiating cells have evolved an elegant mecha- separate metabolic compartment, they are connected to the nucleo- nism coupling new energetic requirements of terminal differentiation to cytosolic compartment through reliance on NMN as a common pre- a change in chromatin composition, setting a higher threshold for cursor of NADþ. However, it is still unclear how exactly NMN enters the PARP1 activation and nuclear NADþ consumption. High levels of mitochondria. Another important question is whether alternative macroH2A1.1 inhibit PARP1, but the exact molecular mechanism of pathways and resources exist that can replenish the mitochondrial this taming remains to be elucidated. NADþ pool. Novel fluorescence-based methods that enable measuring NADþ levels in different compartments in situ will be very helpful to 5. CONCLUSION AND OUTLOOK answer these questions [82]. Future work will need to address the question, which metabolites other than NMN act as signaling mole- Organisms have co-evolved mechanisms governing genome stability, cules between distant compartments? transcriptional regulation, and metabolic homeostasis. It is not sur- A challenge for the PARP field is the development of methods that prising that these mechanisms are interconnected. Shared metabolites enable the detection of low or basal enzymatic activity. The mea- are able to communicate states between distant organelles. It is now surement of PARP-1 activity in cells relies on the detection of its well accepted that NADþ metabolism plays a major role in commu- product, the instable PAR polymer that transiently accumulates after nication between the nucleus and mitochondria [44,45]. While the PARP-1 hyperactivation. Under steady-state conditions, the PAR 6 MOLECULAR METABOLISM 38 (2020) 100950 Ó 2020 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). www.molecularmetabolism.com polymer cannot be detected due to its rapid turnover. While it has been FUNDING convincingly shown that acute activation of PARP1 by DNA damage or other stresses leads to NADþ depletion, much less is known about the Research in the Ladurner and Buschbeck labs was supported by the level of basal NADþ consumption. Our study on myotubes suggested following grants: the Deutsche Forschungsgemeinschaft SFB 646 that even in non-cycling differentiated cells without induction of DNA and SFB 1064 collaborative research centers (to AGL); the Deutsche damage by exogenous agents, basal PARP1 activity exists and if not Forschungsgemeinschaft CIPSM and SyNergy research excellence blunted by macroH2A1.1 can consume a substantial amount of NADþ clusters (to AGL); the Bavarian BioSysNet Program (to AGL); the ERA- [39]. Compensated by the salvage pathway, this was reflected by a NET Neuron project Food4Thought funded by the Bundesministerium depletion of the precursor NMN. The exact consequences of auto-MAR für Bildung und Forschung (to AGL); the Marie Skłodowska Curie and PARylation for PARP activity remain to be studied and will provide a Training network “ChroMe” H2020-MSCA-ITN-2015-675,610 (to MB better understanding of the molecular basis of PARP1 inhibition by and AGL); MINECO RTI2018-094005-B-I00 (to MB); MINECO-ISCIII macroH2A1.1 binding. In this line, it is interesting to note that the linker PIE16/00011 (to MB); the Deutsche José Carreras Leukaemie Stif- of macroH2A1 itself can be ADP-ribosylated on serine 146 [83]. This tung DJCLS 14R/2018 (to MB); AGAUR 2017-SGR-305 (to MB); and leads us to wonder if the linker modification would conversely affect Fundació La Marató de TV3 257/C/2019 (to MB). Research at the IJC macroH2A1.1 capacity to bind and inhibit PARP1. Interestingly, the is supported by the La Caixa Foundation, the Fundació Internacional linker sequences differ between macroH2A1 and macroH2A2. Josep Carreras, Celgene Spain, and the CERCA Program/Generalitat The involvement of macroH2A histone variants in DNA repair is de Catalunya. complex and future research will need to dissect which functions are based on the interplay between macroH2A1.1 and PARP1 and which AUTHOR CONTRIBUTIONS functions are contributions by other macroH2A isoforms and inde- pendent of PARP1. PARP1 activation is an early event in the DNA The literature revision introduction and discussion of the PhD thesis by repair process and its partial inhibition by macroH2A1.1 reduces SHB served as the basis for this review. MB wrote all of the sections PARP1-dependent chromatin expansion [68], avoids DNA damage- related to NADþ metabolism including the section on macroH2A1.1. induced NADþ depletion, and alters the dynamics of PAR by delay- GK and AGL wrote the section on PARP1. The figures were designed by ing its production and increasing its half-life [77]. MacroH2A1.1 is sci.sters.editions (https://sci-sters-editions.com). All the authors edited recruited to PAR [74,84], which can lead to the large-scale reorga- the full manuscript and approved the final version. nization of the chromatin [74] or its local incorporation into the chromatin surrounding the damaged site [76]. As a consequence of CONFLICT OF INTEREST these functions, macroH2A1.1 promotes cell survival in response to ionizing radiation [76] and the repair of oxidative damage [77]. In AGL is a founder and shareholder of Eisbach Bio GmbH. SHB is co-founder of mice, the genetic ablation of macroH2A1.1 led to a reduction of sci.sters.editions. The authors declare no other conflicts of interest. hematopoietic stem cells after high-dose irradiation [85]. Functions more related to later time points of the DNA damage repair process REFERENCES have been reported for macroH2A1.2. 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