The Drosophila gene encoding JIG protein (CG14850) is critical for CrebA nuclear trafficking during development.

Coordination of mitochondrial and nuclear processes is key to the cellular health; however, very little is known about the molecular mechanisms regulating nuclear-mitochondrial crosstalk. Here, we report a novel molecular mechanism controlling the shuttling of CREB (cAMP response element-binding protein) protein complex between mitochondria and nucleoplasm. We show that a previously unknown protein, herein termed as Jig, functions as a tissue-specific and developmental timing-specific coregulator in the CREB pathway. Our results demonstrate that Jig shuttles between mitochondria and nucleoplasm, interacts with CrebA protein and controls its delivery to the nucleus, thus triggering CREB-dependent transcription in nuclear chromatin and mitochondria. Ablating the expression of Jig prevents CrebA from localizing to the nucleoplasm, affecting mitochondrial functioning and morphology and leads to Drosophila developmental arrest at the early third instar larval stage. Together, these results implicate Jig as an essential mediator of nuclear and mitochondrial processes. We also found that Jig belongs to a family of nine similar proteins, each of which has its own tissue- and time-specific expression profile. Thus, our results are the first to describe the molecular mechanism regulating nuclear and mitochondrial processes in a tissue- and time-specific manner.

PARG Protein Regulation Roles in Drosophila Longevity Control.

Aging, marked by a gradual decline in physiological function and heightened vulnerability to age-related diseases, remains a complex biological process with multifaceted regulatory mechanisms. Our study elucidates the critical role of poly(ADP-ribose) glycohydrolase (PARG), responsible for catabolizing poly(ADP-ribose) (pADPr) in the aging process by modulating the expression of age-related genes in Drosophila melanogaster. Specifically, we uncover the regulatory function of the uncharacterized PARG C-terminal domain in controlling PARG activity. Flies lacking this domain exhibit a significantly reduced lifespan compared to wild-type counterparts. Furthermore, we observe progressive dysregulation of age-related gene expression during aging, accelerated in the absence of PARG activity, culminating in a premature aging phenotype. Our findings reveal the critical involvement of the pADPr pathway as a key player in the aging process, highlighting its potential as a therapeutic target for mitigating age-related effects.

Disrupting Poly(ADP-ribosyl)ating Pathway Creates Premalignant Conditions in Mammalian Liver.

Hepatocellular carcinoma (HCC) is a major global health concern, representing one of the leading causes of cancer-related deaths. Despite various treatment options, the prognosis for HCC patients remains poor, emphasizing the need for a deeper understanding of the factors contributing to HCC development. This study investigates the role of poly(ADP-ribosyl)ation in hepatocyte maturation and its impact on hepatobiliary carcinogenesis. A conditional Parg knockout mouse model was employed, utilizing Cre recombinase under the albumin promoter to target Parg depletion specifically in hepatocytes. The disruption of the poly(ADP-ribosyl)ating pathway in hepatocytes affects the early postnatal liver development. The inability of hepatocytes to finish the late maturation step that occurs early after birth causes intensive apoptosis and acute inflammation, resulting in hypertrophic liver tissue with enlarged hepatocytes. Regeneration nodes with proliferative hepatocytes eventually replace the liver tissue and successfully fulfill the liver function. However, early developmental changes predispose these types of liver to develop pathologies, including with a malignant nature, later in life. In a chemically induced liver cancer model, Parg-depleted livers displayed a higher tendency for hepatocellular carcinoma development. This study underscores the critical role of the poly(ADP-ribosyl)ating pathway in hepatocyte maturation and highlights its involvement in liver pathologies and hepatobiliary carcinogenesis. Understanding these processes may provide valuable insights into liver biology and liver-related diseases, including cancer.

PARG suppresses tumorigenesis and downregulates genes controlling angiogenesis, inflammatory response, and immune cell recruitment.

Chemokines are highly expressed in tumor microenvironment and play a critical role in all aspects of tumorigenesis, including the recruitment of tumor-promoting immune cells, activation of cancer-associated fibroblasts, angiogenesis, metastasis, and growth. Poly (ADP-ribose) polymerase (PARP) is a multi-target transcription regulator with high levels of poly(ADP-ribose) (pADPr) being reported in a variety of cancers. Furthermore, poly (ADP-ribose) glycohydrolase (PARG), an enzyme that degrades pADPr, has been reported to be downregulated in tumor tissues with abnormally high levels of pADPr. In conjunction to this, we have recently reported that the reduction of pADPr, by either pharmacological inhibition of PARP or PARG's overexpression, disrupts renal carcinoma cell malignancy in vitro. Here, we use 3 T3 mouse embryonic fibroblasts, a universal model for malignant transformation, to follow the effect of PARG upregulation on cells' tumorigenicity in vivo. We found that the overexpression of PARG in mouse allografts produces significantly smaller tumors with a delay in tumor onset. As downregulation of PARG has also been implicated in promoting the activation of pro-inflammatory genes, we also followed the gene expression profile of PARG-overexpressing 3 T3 cells using RNA-seq approach and observed that chemokine transcripts are significantly reduced in those cells. Our data suggest that the upregulation of PARG may be potentially useful for the tumor growth inhibition in cancer treatment and as anti-inflammatory intervention.

Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly(ADP-ribosyl)ation.

Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes.

Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, we demonstrate that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This "hit and run" activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites.

Charon Mediates Immune Deficiency-Driven PARP-1-Dependent Immune Responses in Drosophila.

Regulation of NF-κB nuclear translocation and stability is central to mounting an effective innate immune response. In this article, we describe a novel molecular mechanism controlling NF-κB-dependent innate immune response. We show that a previously unknown protein, termed as Charon, functions as a regulator of antibacterial and antifungal immune defense in Drosophila Charon is an ankyrin repeat-containing protein that mediates poly(ADP-ribose) polymerase-1 (PARP-1)-dependent transcriptional responses downstream of the innate immune pathway. Our results demonstrate that Charon interacts with the NF-κB ortholog Relish inside perinuclear particles and delivers active Relish to PARP-1-bearing promoters, thus triggering NF-κB/PARP-1-dependent transcription of antimicrobial peptides. Ablating the expression of Charon prevents Relish from targeting promoters of antimicrobial genes and effectively suppresses the innate immune transcriptional response. Taken together, these results implicate Charon as an essential mediator of PARP-1-dependent transcription in the innate immune pathway. Thus, to our knowledge, our results are the first to describe the molecular mechanism regulating translocation of the NF-κB subunit from cytoplasm to chromatin.

Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA.

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.

Bookmarking promoters in mitotic chromatin: poly(ADP-ribose)polymerase-1 as an epigenetic mark.

Epigenetics are the heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. After mitosis, it is thought that bookmarking transcription factors remain at promoters, regulating which genes become active and which remain silent. Herein, we demonstrate that poly(ADP-ribose)polymerase-1 (PARP-1) is a genome-wide epigenetic memory mark in mitotic chromatin, and we further show that the presence of PARP-1 is absolutely crucial for reactivation of transcription after mitosis. Based on these findings, a novel molecular model of epigenetic memory transmission through the cell cycle is proposed.

Poly(ADP-Ribose) polymerase 1 (PARP-1) regulates ribosomal biogenesis in Drosophila nucleoli.

Poly(ADP-ribose) polymerase 1 (PARP1), a nuclear protein, utilizes NAD to synthesize poly(AD-Pribose) (pADPr), resulting in both automodification and the modification of acceptor proteins. Substantial amounts of PARP1 and pADPr (up to 50%) are localized to the nucleolus, a subnuclear organelle known as a region for ribosome biogenesis and maturation. At present, the functional significance of PARP1 protein inside the nucleolus remains unclear. Using PARP1 mutants, we investigated the function of PARP1, pADPr, and PARP1-interacting proteins in the maintenance of nucleolus structure and functions. Our analysis shows that disruption of PARP1 enzymatic activity caused nucleolar disintegration and aberrant localization of nucleolar-specific proteins. Additionally, PARP1 mutants have increased accumulation of rRNA intermediates and a decrease in ribosome levels. Together, our data suggests that PARP1 enzymatic activity is required for targeting nucleolar proteins to the proximity of precursor rRNA; hence, PARP1 controls precursor rRNA processing, post-transcriptional modification, and pre-ribosome assembly. Based on these findings, we propose a model that explains how PARP1 activity impacts nucleolar functions and, consequently, ribosomal biogenesis.

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. Here, we identify histone variants that regulate the targeting and enzymatic activity of poly(ADP-ribose) polymerase 1 (PARP1), a chromatin regulator in higher eukaryotes. We demonstrate that 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 that 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. Our 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.

Uncoupling of the transactivation and transrepression functions of PARP1 protein.

Poly(ADP ribose) polymerase 1 (PARP1) is a nuclear protein that regulates chromatin remodeling and transcription as well as DNA repair and genome stability pathways. Recent studies have revealed a paradoxical dual role of PARP1 protein in transcription. Specifically, although PARP1 controls transcriptional activation of a subset of genes that are heat shock- or hormone-dependent, it also directly inactivates transcription, establishes heterochromatin domains, and silences retrotransposable elements. However, the domains required for these disparate functions are currently unknown. In this paper, we report the discovery of a previously undescribed mutation in the Drosophila Parp locus. We show that the mutants express a deletion mutant of PARP1 protein with an altered DNA binding domain that carries only the second Zn-finger. We demonstrate that this alteration specifically excludes PARP1 protein from heterochromatin and makes PARP1 unable to maintain repression of retrotransposable elements. By characterizing the biological activity of this unique PARP1 mutant protein isoform, we have uncoupled the transactivation and transrepression functions of this protein.

Post-transcriptional regulation by poly(ADP-ribosyl)ation of the RNA-binding proteins.

Gene expression is intricately regulated at the post-transcriptional level by RNA-binding proteins (RBPs) via their interactions with pre-messenger RNA (pre-mRNA) and mRNA during development. However, very little is known about the mechanism regulating RBP activities in RNA metabolism. During the past few years, a large body of evidence has suggested that many RBPs, such as heterogeneous nuclear ribonucleoproteins (hnRNPs), undergo post-translational modification through poly(ADP-ribosyl)ation to modulate RNA processing, including splicing, polyadenylation, translation, miRNA biogenesis and rRNA processing. Accordingly, RBP poly(ADP-ribosyl)ation has been shown to be involved in stress responses, stem cell differentiation and retinal morphogenesis. Here, we summarize recent advances in understanding the biological roles of RBP poly(ADP-ribosyl)ation, as controlled by Poly(ADP-ribose) Polymerases (PARPs) and Poly(ADP-ribose) Glycohydrolase (PARG). In addition, we discuss the potential of PARP and PARG inhibitors for the treatment of RBP-related human diseases, including cancer and neurodegenerative disorders.

TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.

Members of PARP family are responsible for poly(ADP-ribose) (pADPr) posttranslational modification synthesis. They are intensively studied proteins with more than 20,500 related papers in PubMed database search to date. PARG, the main enzyme that degrades pADPr, is unfairly attracted less attention, and 40 times less papers (a little more than 500) are related to its functioning. The difficulties to work with PARG knockout animals due to its early embryo lethality could be one reason for this huge difference. Mice PARG-specific antibodies are not available from any vendor, which also complicates the research process. There is one available for public PARG knockout mice line generated by KOMP project. It has LacZ cassette, which replaces three critical exons in PARG gene. Here, we present the method to genotype these mice with Taqman qPCR multiplex approach. It allows to work with a small amount of DNA material like early embryo stages and to separate maternal DNA contamination. The modification of this method is also applicable for studying PARG conditional knockouts and identifying the success of floxed PARG gene exon deletion by Cre-driven recombination.

Generating PARP Knockout D. melanogaster with CRISPR/Cas9 System.

Long-branched negatively charged poly(ADP-ribose) (pADPr) is a posttranslation modification of nuclear proteins that play a key role in many chromatin remodeling events. While several enzymes of PARP family could synthesize it across all multicellular organisms, Drosophila melanogaster is very suitable model to study pADPr-regulated processes because only one PARP gene is present. Despite the fact that PARP is an intensively studied protein with multiple important functions, no total knockout PARP flies were obtained in mobile element mutagenesis-based projects, mainly because PARP gene localizes in heterochromatic region. Here, we describe all steps of generating PARP mutated D. melanogaster with CRISPR/Cas9 system from the gRNA design, plasmid cloning to fly crosses and mutation detection. Provided gRNAs sequences target the region with high efficiency and results in more than 90% mutant stocks. This method could also be modified to generate PARP mutations in other gene locus, knockins with donor sequences for homology recombination or to be adjusted for other pADPr turnover-regulating enzymes.

Using Drosophila Genetics to Identify Factors that Affect PARP1 Activity In Vivo.

PARP1 is the enzyme responsible for the majority of the poly(ADP-ribose) (pADPr) synthesis in Drosophila. Its activity can be easily evaluated in vitro by measuring the level of pADPr, which allow to study the effect of potential PARP1 upstream factors on PARP1 activity. However, PARP1 activity can be challenging to measure in vivo, due to the presence of PARG, since pADPr level is a consequence of the activity of both PARP1 that synthetizes pADPr and PARG that degrades it. An increase in PARG activity can hide an increase of PARP1 activity. In this context, the effect of potential upstream factors on PARP1 activity can be hard to measure. Here, we describe a genetic background where PARG is absent to study changes in PARP1 activity at different developmental time points.

Cell-Based Screening for New PARP Inhibitors Utilizing PARG-Mutated Mouse Embryonic Stem Cells.

According to the most recent data, cancer is among the leading cause of death in the United States and accounted for more than 600,000 deaths in 2021. Around 30% of these cancer-related deaths were caused by breast, prostate, and ovarian cancers. PARP-1 inhibitors show the most promising results in treatment of these three types of cancers and have found widespread use in the development of novel treatment strategies. A number of PARP inhibitors currently are undergoing phase I/II of FDA approval process for treatment of genetically disposed mutant tumors. Recently, however, a few clinical studies reported setbacks in research on PARP-1 inhibitors. It is likely that these setbacks are caused by tremendous off-target effects. To overcome these problems, it is very important to design new potent PARP-1 inhibitors, which do not kill normal cells. Our newly developed assay is based on the usage of sensitized embryonic stem cells with disrupted PARG gene that significantly increase the base level of pADPr for easy detection. Our approach allows the discovery of that effectively target poly(ADP-ribosyl)ation in cells and allows to select compounds with minimal or no cytotoxic effects on ES cells.

Poly(ADP-ribosyl)ating enzymes coordinate changes in the expression of metabolic genes with developmental progression.

Metabolism, known to be temporally regulated to meet evolving energy demands, plays a crucial role in shaping developmental pace. Recent studies have demonstrated that two key proteins PARP1 and PARG play a regulatory role in the transcription of both morphogenic and metabolic genes. Intriguingly, in Drosophila, the depletion of PARP1 or PARG proteins causes a developmental arrest before pupation, resulting in individuals unable to complete their development. This phenotype highlights the critical involvement of poly(ADP-ribosyl)ating enzymes in regulating the metamorphic process. In this study, we provide compelling evidence that these enzymes intricately coordinate transcriptional changes in both developmental and metabolic pathways during metamorphosis. Specifically, they promote the expression of genes crucial for pupation, while simultaneously negatively regulating the expression of metabolic genes before the transition to the pupal stage. Additionally, these enzymes suppress the expression of genes that are no longer required during this transformative period. Our findings shed light on the intricate interplay between poly(ADP-ribosyl)ating enzymes, developmental processes, and metabolic regulation before metamorphosis and highlight a new role of poly(ADP-ribosyl)ating enzymes in the global regulation of transcription.

Chromatin Immunoprecipitation Approach to Determine How PARP1 Domains Affect Binding Pattern to Chromatin.

Poly(ADP-ribose) polymerase 1 (PARP1) is an enzyme involved in the regulation of different cellular mechanisms, ranging from DNA repair to regulation of gene expression. The different PARP1 domains have been shown to influence PARP1 binding pattern to chromatin. However, which loci bound by PARP1 are affected in the absence of a specific domain is not known. To determine the binding pattern of the different PARP1 domains, we used a ChIP-seq approach on different GFP-tagged versions of PARP1. Here, we described how to perform and analyze ChIP-seq performed with a GFP antibody in Drosophila melanogaster third instar larvae.

Poly (ADP-ribose) polymerase 1 is required for protein localization to Cajal body.

Recently, the nuclear protein known as Poly (ADP-ribose) Polymerase1 (PARP1) was shown to play a key role in regulating transcription of a number of genes and controlling the nuclear sub-organelle nucleolus. PARP1 enzyme is known to catalyze the transfer of ADP-ribose to a variety of nuclear proteins. At present, however, while we do know that the main acceptor for pADPr in vivo is PARP1 protein itself, by PARP1 automodification, the significance of PARP1 automodification for in vivo processes is not clear. Therefore, we investigated the roles of PARP1 auto ADP-ribosylation in dynamic nuclear processes during development. Specifically, we discovered that PARP1 automodification is required for shuttling key proteins into Cajal body (CB) by protein non-covalent interaction with pADPr in vivo. We hypothesize that PARP1 protein shuttling follows a chain of events whereby, first, most unmodified PARP1 protein molecules bind to chromatin and accumulate in nucleoli, but then, second, upon automodification with poly(ADP-ribose), PARP1 interacts non-covalently with a number of nuclear proteins such that the resulting protein-pADPr complex dissociates from chromatin into CB.