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.

Mono-methylated histones control PARP-1 in chromatin and transcription.

PARP-1 is central to transcriptional regulation under both normal and stress conditions, with the governing mechanisms yet to be fully understood. Our biochemical and ChIP-seq-based analyses showed that PARP-1 binds specifically to active histone marks, particularly H4K20me1. We found that H4K20me1 plays a critical role in facilitating PARP-1 binding and the regulation of PARP-1-dependent loci during both development and heat shock stress. Here, we report that the sole H4K20 mono-methylase, pr-set7, and parp-1 Drosophila mutants undergo developmental arrest. RNA-seq analysis showed an absolute correlation between PR-SET7- and PARP-1-dependent loci expression, confirming co-regulation during developmental phases. PARP-1 and PR-SET7 are both essential for activating hsp70 and other heat shock genes during heat stress, with a notable increase of H4K20me1 at their gene body. Mutating pr-set7 disrupts monomethylation of H4K20 along heat shock loci and abolish PARP-1 binding there. These data strongly suggest that H4 monomethylation is a key triggering point in PARP-1 dependent processes in chromatin.

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.

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.

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.

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.

Poly(ADP-ribosyl)ating enzymes cooperate to coordinate development.

The transcriptome is subject to rapid and massive changes during the transition between developmental stages. These changes require tight control to avoid the undesired reactivation of gene expression that is only important for previous developmental stages and, if unchecked during transition between developmental stages, could lead to anarchic proliferation and formation of malignant tumors. In this context, the involvement of chromatin factors is important since they can directly regulate the expression of multiple genes at the same time. Poly(ADP-ribose) enzymes, involved in several processes from DNA repair to transcription regulation, might play a role in this regulation. Here, we report that PARP-1 and PARG cooperate to temporally regulate the gene expression profile during the larval/pupa transition. PARP-1 and PARG are both essential in repressing the expression of genes coding for digestive enzymes and larval cuticle proteins, while PARG positively regulate the expression of defense response genes. These results suggest a cooperative coordination between PARP-1 and PARG that specifically maintains the integrity of expression profile between developmental stages.

Upregulation of PARG in prostate cancer cells suppresses their malignant behavior and downregulates tumor-promoting genes.

Post-translational modification of nuclear proteins through the addition of poly(ADP-ribose) (pADPr) moieties is upregulated in many metastatic cancers, where the high levels of pADPr have often been associated with poor cancer prognosis. Although the inhibitors of poly(ADP-ribose) polymerases (PARPs) have been utilized as potent anti-cancer agents, their efficacy in clinical trials varied among patient groups and has often been unpredictable. Such outcome cannot be interpreted solely by the inability to keep PARP-driven DNA repair in check. The focus of studies on PARP-driven tumorigenesis have recently been shifted toward PARP-dependent regulation of transcription. Here we utilized the controlled overexpression of poly(ADP-ribose) glycohydrolase (PARG), a sole pADPr-degrading enzyme, to investigate pADPr-dependent gene regulation in prostate cancer PC-3 cells. We demonstrated that PARG upregulation reduces pADPr levels and inhibits the expression of genes in key tumor-promoted pathways, including TNFα/NF-kB, IL6/STAT3, MYC, and KRAS signaling, the genes involved in inflammation response, especially chemokines, and endothelial-mesenchymal transition. The observed effect of PARG on transcription was consistent across all tested prostate cancer cell lines and correlates with PARG-induced reduction of clonogenic potential of PC-3 cells in vitro and a significant growth inhibition of PC-3-derived tumors in nude mice in vivo.

PRC1 chromatin factors strengthen the consistency of neuronal cell fate specification and maintenance in C. elegans.

In the nervous system, the specific identity of a neuron is established and maintained by terminal selector transcription factors that directly activate large batteries of terminal differentiation genes and positively regulate their own expression via feedback loops. However, how this is achieved in a reliable manner despite noise in gene expression, genetic variability or environmental perturbations remains poorly understood. We addressed this question using the AIY cholinergic interneurons of C. elegans, whose specification and differentiation network is well characterized. Via a genetic screen, we found that a loss of function of PRC1 chromatin factors induces a stochastic loss of AIY differentiated state in a small proportion of the population. PRC1 factors act directly in the AIY neuron and independently of PRC2 factors. By quantifying mRNA and protein levels of terminal selector transcription factors in single neurons, using smFISH and CRISPR tagging, we observed that, in PRC1 mutants, terminal selector expression is still initiated during embryonic development but the level is reduced, and expression is subsequently lost in a stochastic manner during maintenance phase in part of the population. We also observed variability in the level of expression of terminal selectors in wild type animals and, using correlation analysis, established that this noise comes from both intrinsic and extrinsic sources. Finally, we found that PRC1 factors increase the resistance of AIY neuron fate to environmental stress, and also secure the terminal differentiation of other neuron types. We propose that PRC1 factors contribute to the consistency of neuronal cell fate specification and maintenance by protecting neurons against noise and perturbations in their differentiation program.

Poly(ADP-ribosyl)ating pathway regulates development from stem cell niche to longevity control.

The regulation of poly(ADP-ribose) polymerase, the enzyme responsible for the synthesis of homopolymer ADP-ribose chains on nuclear proteins, has been extensively studied over the last decades for its involvement in tumorigenesis processes. However, the regulation of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for removing this posttranslational modification, has attracted little attention. Here we identified that PARG activity is partly regulated by two phosphorylation sites, ph1 and ph2, in Drosophila We showed that the disruption of these sites affects the germline stem-cells maintenance/differentiation balance as well as embryonic and larval development, but also the synchronization of egg production with the availability of a calorically sufficient food source. Moreover, these PARG phosphorylation sites play an essential role in the control of fly survivability from larvae to adults. We also showed that PARG is phosphorylated by casein kinase 2 and that this phosphorylation seems to protect PARG protein against degradation in vivo. Taken together, these results suggest that the regulation of PARG protein activity plays a crucial role in the control of several developmental processes.

Age-Related Changes of Gene Expression Profiles in Drosophila.

An individual's gene expression profile changes throughout their life. This change in gene expression is shaped by differences in physiological needs and functions between the younger and older organism. Despite intensive studies, the aging process is not fully understood, and several genes involved in this process may remain to be identified. Here we report a transcriptomic analysis of Drosophila melanogaster using microarrays. We compared the expression profiles of two-day-old female adult flies with those of 45-day-old flies. We identified 1184 genes with pronounced differences in expression level between young and old age groups. Most genes involved in muscle development/maintenance that display different levels of expression with age were downregulated in older flies. Many of these genes contributed to sarcomere formation and function. Several of these genes were functionally related to direct and indirect flight muscles; some of them were exclusively expressed in these muscles. Conversely, several genes involved in apoptosis processes were upregulated in aging flies. In addition, several genes involved in resistance to toxic chemicals were upregulated in aging flies, which is consistent with a global upregulation of the defense response system in aging flies. Finally, we randomly selected 12 genes among 232 genes with unknown function and generated transgenic flies expressing recombinant proteins fused with GFP protein to determine their subcellular expression. We also found that the knockdown of some of those 12 genes can affect the lifespan of flies.

Poly(ADP-ribose) polymerase 1 in genome-wide expression control in Drosophila.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a nuclear enzyme involved in DNA repair and transcription regulation, among other processes. Malignant transformations, tumor progression, the onset of some neuropathies and other disorders have been linked to misregulation of PARP-1 activity. Despite intensive studies during the last few decades, the role of PARP-1 in transcription regulation is still not well understood. In this study, a transcriptomic analysis in Drosophila melanogaster third instar larvae was carried out. A total of 602 genes were identified, showing large-scale changes in their expression levels in the absence of PARP-1 in vivo. Among these genes, several functional gene groups were present, including transcription factors and cytochrome family members. The transcription levels of genes from the same functional group were affected by the absence of PARP-1 in a similar manner. In the absence of PARP-1, all misregulated genes coding for transcription factors were downregulated, whereas all genes coding for members of the cytochrome P450 family were upregulated. The cytochrome P450 proteins contain heme as a cofactor and are involved in oxidoreduction. Significant changes were also observed in the expression of several mobile elements in the absence of PARP-1, suggesting that PARP-1 may be involved in regulating the expression of mobile elements.

Wnt ligands regulate the asymmetric divisions of neuronal progenitors in C. elegans embryos.

Wnt/ß-catenin signalling has been implicated in the terminal asymmetric divisions of neuronal progenitors in vertebrates and invertebrates. However, the role of Wnt ligands in this process remains poorly characterized. Here, we used the terminal divisions of the embryonic neuronal progenitors in C. elegans to characterize the role of Wnt ligands during this process, focusing on a lineage that produces the cholinergic interneuron AIY. We observed that, during interphase, the neuronal progenitor is elongated along the anteroposterior axis, then divides along its major axis, generating an anterior and a posterior daughter with different fates. Using time-controlled perturbations, we show that three Wnt ligands, which are transcribed at higher levels at the posterior of the embryo, regulate the orientation of the neuronal progenitor and its asymmetric division. We also identify a role for a Wnt receptor (MOM-5) and a cortical transducer APC (APR-1), which are, respectively, enriched at the posterior and anterior poles of the neuronal progenitor. Our study establishes a role for Wnt ligands in the regulation of the shape and terminal asymmetric divisions of neuronal progenitors, and identifies downstream components.

Zic Genes in Nematodes: A Role in Nervous System Development and Wnt Signaling.

Transcription factors of the Zic family play important roles during animal development, and their misregulation has been implicated in several human diseases. Zic proteins are present in nematodes, and their function has been mostly studied in the model organism C. elegans. C. elegans possesses only one Zic family member, REF-2. Functional studies have shown that this factor plays a key role during the development of the nervous system, epidermis, and excretory system. In addition, they have revealed that the C. elegans Zic protein acts as an atypical mediator of the Wnt/ß-catenin pathway. In other animals including vertebrates, Zic factors are also regulators of nervous system development and modulators of Wnt signaling, suggesting that these are evolutionary ancient functions of Zic proteins.

Independent synchronized control and visualization of interactions between living cells and organisms.

To investigate the early stages of cell-cell interactions occurring between living biological samples, imaging methods with appropriate spatiotemporal resolution are required. Among the techniques currently available, those based on optical trapping are promising. Methods to image trapped objects, however, in general suffer from a lack of three-dimensional resolution, due to technical constraints. Here, we have developed an original setup comprising two independent modules: holographic optical tweezers, which offer a versatile and precise way to move multiple objects simultaneously but independently, and a confocal microscope that provides fast three-dimensional image acquisition. The optical decoupling of these two modules through the same objective gives users the possibility to easily investigate very early steps in biological interactions. We illustrate the potential of this setup with an analysis of infection by the fungus Drechmeria coniospora of different developmental stages of Caenorhabditis elegans. This has allowed us to identify specific areas on the nematode's surface where fungal spores adhere preferentially. We also quantified this adhesion process for different mutant nematode strains, and thereby derive insights into the host factors that mediate fungal spore adhesion.