LAUSR

Poly(ADP-ribosylation) (PARylation) is a reversible post-translational protein modification that regulates a number of cellular processes. It is generated by a family of PAR polymerases (PARP), of which PARP1 has recently been exploited as a target in ovarian cancer carrying BRCA1/2 mutations. In addition, tankyrase (PARP5A and B), with its regulation of Wnt signalling, telomeres and the mitotic apparatus, has attracted much effort in the development of targeted therapies. All this necessitates the identification of PARylation targets and their mode of regulation by the modification. Genetically encoded fluorescent biosensor that can report the PARylation state of a target protein has the potential to report the modulation of the PARylation target in living cells. We used PAR-binding PBZ domains fused to a non-fluorescent fragment of a GFP molecule in combination with a protein of interest fused to the complementing fragment of GFP. When this protein is PARylated, it is recognized by the PBZ domain, which brings in proximity the two GFP fragments, and restores the GFP fluorescence. Known targets of PARylation, e.g. TERF1 and AXIN1, showed specific spatial patterns of PARylation validating our approach. In order to identify novel PARylation targets, we have designed a genetic screen based on the biosensor. We introduced the GFP fragment in a transposon, which was randomly integrated in cellular genome, thus generating cellular libraries of thousands of tagged genes. The introduction of the PAR binding probe lead to GFP fluorescence in clones that harbour putative PARylation targets. We developed a next generation sequencing approach to isolate the transposon integration sites and annotated the target genes. Reassuringly, we have identified NPM1, a well-known PARylation target, as a strongly scoring gene. We focused on one of novel identified hits (CTIF). We showed that CTIF is PARylated in vivo in a tankyrase-dependent manner. Furthermore, the biosensor revealed that this PARylation is predominant in a specific cellular compartment surrounding the centrosome. Functional studies have implicated this protein the regulation of the centrosomal satellites. Collectively, we are presenting a new approach to identify PARylated proteins and study their patterns of PARylation and regulation in vivo. Citation Format: Dragomir B. Krastev, Stephen J. Pettitt, James Campbell, Alan Ashworth, Christopher J. Lord. A PARylation biosensor genetic screen to identify novel PARylation targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1360.