PARG in Repair, Replication And Transcription – PIRRAT

The lack of bioavailable PARG inhibitors, the presence of numerous isoforms and the lethality of the complete PARG mouse knockout are obstacles to tackle the function of PARG. We are therefore generating new human and mouse cellular models deficient in endogenous PARG by knock out or stable shRNA expression, and complemented with single PARG isoforms or PARG mutants defective for binding to protein partners, such as PCNA. These models will allow us to explore the role or each PARG isoform and their interaction with PCNA in DNA repair, DNA replication and transcription. We expect that thanks to the proposed experiments we will gain significant knowledge on the function of PARG in response to replicative stress and DNA damage, transcription and cell differentiation as well on the cellular roles of the different PARG isoforms, which are largely understudied.

Task 2 is almost achieved: we have shown that PARG-deprived cells display an enhanced sensitivity to the replication inhibitor hydroxyurea and that PARG is dispensable to recover from transient replicative stress but is necessary to avoid massive PAR production upon prolonged replicative stress, conditions leading to fork collapse and DSB. Extensive PAR accumulation impairs replication protein A (RPA) association with collapsed forks, resulting in compromised DSB repair via homologous recombination. Our results highlight the critical role of PARG in tightly controlling PAR levels produced upon genotoxic stress to prevent the detrimental effects of PAR over-accumulation. These results are published in Illuzzi et al, 2014, Nucleic Acids Res, 42, 7776-92. For tasks 3 and 4, we are about to obtain the cellular models deficient in endogenous PARG and complemented with PARG isoforms and PARG mutants.

Our project is at the forefront of on-going international studies aimed to dissect the biochemical and functional aspects of poly(ADP-ribosyl)ation. This is of particular importance knowing that PARP inhibitors have entered clinical trials for their promising applications for cancer therapy and treatment of various inflammatory or neuropathic diseases. PARG inhibitors, despite that the few available are currently not satisfying for cellular and animal uses, are also endowed with an increasing interest for at least cancer therapy. It is therefore essential to better characterize the biological role of PARG and precisely define the consequences of an impaired PARG activity. Due to the lack of bioavailable PARG inhibitors, the presence of numerous isoforms and the lethality of the complete PARG knock out, the shRNA strategy to knock down in cells all PARG isoforms and subsequent complementation with single isoforms or mutants is a powerful strategy to tackle the function of PARG in DNA repair, replicative stress or transcription, with now a possible access to the contribution of each PARG isoforms and of the interplay with PCNA in all these processes.

PARG is dispensable for recovery from transient replicative stress but required to prevent detrimental accumulation of poly(ADP-ribose) upon prolonged replicative stress.
Illuzzi G, Fouquerel E, Amé JC, Noll A, Rehmet K, Nasheuer HP, Dantzer F and Schreiber V. (2014). Nucleic Acids Res. 42, 7776-92.

Poly(ADP-ribosyl)ation (PARylation) is a post-translational modification of proteins mediated by Poly(ADP-ribose) polymerases (PARPs), which catalyse the transfer and polymerisation of ADP-ribose units from NAD+ to form a ramified poly(ADP-ribose) polymer (PAR), covalently linked to acceptor proteins. PARylation was initially described as a survival event in the cell response to DNA damages, to accelerate repair of DNA breaks. The PAR cellular level is regulated by PARPs and the degrading enzyme poly(ADP-ribose) glycohydrolase (PARG), influencing the cell fate decision between life and death in response to DNA damage. Besides its critical role in the maintenance of genome integrity, PARylation was shown to play essential role in various cellular processes including transcriptional regulation, organisation of chromatin domains, cell cycle progression or cell differentiation. Whereas the role of the founding member of the PARP family PARP-1 is highly documented in these processes, far less is known about PARG. In contrast to the 17 different PARP genes, there is 1 PARG gene that encodes several isoforms displaying various subcellular localizations: nuclear, cytoplasmic and mitochondrial. The relative role of each isoforms is not elucidated yet, but their existence and the lethality of a complete PARG knockout in mice have complicated the study of PARG biological and physiological function. It is however becoming clear that despite having antagonistic activities on PAR metabolism, PARP-1 and PARG do not necessarily display antagonistic functions in cells. It is therefore essential to better understand how PARG contributes to the dynamic regulation of PARylation to ensure the tight control of the cellular processes this modification is involved in.

In this context, significative advances have already been made by the partners of this project: Partner 1 has previously shown that the absence of PARG increased radiosensitivity and affected the repair of radioinduced single and double strand breaks (Amé et al, 2009, J. Cell Sci. 122, 1990-2002). Partner 1 also revealed a functional link between PARG and the repair/replication factor PCNA, required for efficient localization of PARG to DNA damage sites and to replication foci (Mortusewicz et al, 2011, Nucleic Acids Res. 39, 5045-56). Finally, Partner 1 and Partner 2 have recently demonstrated the crucial role of PARG in the transactivation of genes mediated by retinoic acid receptor (Le May et al, 2012, Mol Cell 48, 785-98).

The present project is aimed to provide an in depth functional characterization of the role of PARG and PARylation in repair, transcription and replication. Partners 1 and 2 will generate new original cellular models that, combined with their expertise in the study of DNA repair, replication and transcription processes, will allow them to:
- determine the role of PARG in cell response to replicative stress (Partner 1)
- determine the role of PARG in transcription regulation in the well-established transcriptional reprograming of mouse ES cell into neurones upon treatment with retinoic acid (Partner 2).
- characterize the functional interplay between PARG and PCNA and determine its eventual implication in repair, replication or transcription (Partner 1)

The present project will provide fundamental knowledge on the role of the major PAR-degrading enzyme, PARG, still poorly documented, in repair, transcription and replication.