Characterization of PARP-3 in genomic stability and epithelial plasticity. – PARP-3

To explore the biochemical properties of the protein, we express the protein in large amounts in insect cells and purify it. Its enzymatic properties (synthesis of poly (ADP-ribose)) are studied in vitro. Protein interactions are identified by immunoprecipitation of the protein using a specific antibody and determination of the protein partners by mass spectrometry. To evaluate the functional properties of PARP-3, we generated a loss of function mouse model in which the protein is not expressed, and we analyse the phenotype. We also use human cell lines in which PARP-3 expression is depleted using siRNA aproches and we explore the consequences of this depletion on a set of cellular processes.

Within the questions already developed, we have identified PARP-3 as a new player in cell response to double strand breaks and mitotic progression. PARP-3 is recruited to sites of DNA damage. Its absence sensitizes human cells to ionizing radiation and causes a significant delay in mitotic division. Our recent results define PARP-3 as a new actor in double-strand break repair by nonhomologous end-joining.
We have also demonstrated an important role of this protein in the repair of physiological strand breaks induced during lymphocyte maturation. Finally, initial data suggest a role of this protein in epithelial plasticity. Together, our results introduce PARP-3 as an important player in tumorigenesis and suggest that its inhibition might be of therapeutic benefit.

We continue this work by dissecting the role of PARP-3 in non-homologous end-joining. We study the consequences of its absence in lymphocyte maturation and especially V(D)J recombination. We explore the impact of its expression in epithelial plasticity. Finally, we study the consequences of its depletion in human tumor cell lines.

Our current results defining PARP-3 as a new player in cell response to double-strand breaks and cell division (mitosis) have been published in PNAS (2011), Cell Cycle (2011) and as a brief «Live from the Labs« at the CNRS.

Poly(ADP-ribosyl)ation 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. DNA breaks are considered to be the major trigger of PAR synthesis through the activation of the founding member PARP-1, favoring DNA repair. PARP-1 inhibitors appeared to have promising pharmacological applications, to potentialize the antitumor effect of chimio- and radiotherapies (phase 1 to 3 clinical trials in progress). The recent discovery of a PARP family (17 members) and of inducers of PAR synthesis other than DNA strand breaks underline the potential of this molecule to control an increasing number of various biological functions including the cellular response to DNA damage, transcriptional regulation, cell cycle progression, cell differentiation, cell death, intracellular transport, lipid metabolism and tumorigenesis [1, 2]. In addition, recent studies revealed that whereas some PARP family members catalyze poly(ADP-ribosyl)ation, others function as mono(ADP-ribosyl)transferase [3]. Together, these findings support the necessity to biochemically and physiologically define the properties of individual PARPs.
By combining biochemical, structural and genetic approaches, our laboratory has extensively contributed to describe PARP-1 and PARP-2 as active players of the base excision repair/single strand break repair (BER/SSBR) process and have discovered both cooperative and more specific functions of both enzymes in heterochromatin integrity.
Recently, we and others have identified PARP-3 as another DNA-dependent PARP but its enzymatic and functional characterization is still poorly documented [4, 5]. The initial studies identified a centrosomal PARP-3 involved in cell cycle progression and a nuclear isoform involved in transcriptional silencing and in the cellular response to DNA damage [4, 6]. Our preliminary cellular evidences indicate a contribution of PARP-3 in the maintenance of genome integrity and identify a significant increased expression of PARP-3 during the processes of epithelial to mesenchymal transition (EMT), two hallmarks of cancer biology. In this proposal, we combine three distinct expertises to determine the biochemical specifics and functional properties of PARP-3 in cellular respose to DNA damage and in epithelial plasticity. By in vitro approaches, we will identify the DNA structures recognized by and activating PARP-3, define its enzymatic properties and identify its partners. We will generate and characterize PARP-3-deficient cellular and mouse models to explore its role in cellular response to DNA damage and in cell division. We have engineered a PARP-3 depleted breast cancer cell line to examine its contribution in epithelial to mesenchymal transition and in cancer invasiveness. Altogether, we believe that our results will define whether PARP-3, like PARP-1, can constitute a novel possible therapeutic target for cancer treatment.

[1] A. Hakme et al. EMBO reports 9 (2008) 1094-1100.
[2] V. Schreiber et al. Nat Rev Mol Cell Biol 7 (2006) 517-528.
[3] H. Kleine et al. Mol Cell 32 (2008) 57-69.
[4] A. Augustin et al. J Cell Sci 116 (2003) 1551-1562.
[5] M. Johansson et al. Genomics 57 (1999) 442-445.
[6] M. Rouleau et al. J Cell Biochem 100 (2007) 385-401.