Parp3 promotes long-range end-joining in murine cells

Abstract

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical-and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.. CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under aThe copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/255281 doi: bioRxiv preprint first posted online Jan. 28, 2018; Chromosomal rearrangements are critical events in the pathogenesis of malignant and nonmalignant disorders (1-3). These aberrant events drive malignant transformation and congenital disorders, including deafness, schizophrenia, and infertility. Many efforts to elucidate the genetic basis of rearrangement formation have relied upon experiments in mouse cells: studies in murine embryonic stem cells (mESCs) identified a cohort of genetic factors that promote or suppress rearrangements (4-8). In aggregate, these studies suggest that chromosomal rearrangements form by a non-canonical or alternative non-homologous end joining pathway (alt-NHEJ). However, a subsequent report demonstrated that in human cells, rearrangement between endonuclease-induced double-strand breaks (DSBs) depends on classical NHEJ (c-NHEJ) (9). The disparate results suggest that the genetic basis for rearrangements differs significantly between human and murine cells.We recently reported that PARP3, a member of the ADP-Ribose Polymerase family of enzymes, promotes chromosomal rearrangement formation in human cells (10). We showed that PARP3 regulates G quadruplex (G4) DNA in response to DNA damage. Chemical stabilization of G4 DNA in PARP3 -/-cells led to widespread DSBs. This suggested a model in which PARP3 suppresses G4 DNA, which allows for processing of DSB ends to intermediates that participate in rearrangements in huma...

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“…Poly(ADP-ribose) polymerase 3 (PARP3) is a member of the PARP protein family that catalyzes mono-ADPribosylation (MARylation), the addition of a single ADPribose unit onto its protein or DNA targets. PARP3 has been characterized for its functions in the repair of DNA double-strand breaks via non homologous endjoining [16][17][18][19] , in the ADP-ribosylation of chromatin at site specific single strand breaks [20][21][22] , in chromosome rearrangements 23,24 , in mitotic segregation 17 and in transcriptional regulation in the zebrafish 25 . More recently, PARP3 has also been defined for its contribution in tumor aggressiveness exemplifying its selective inhibition as an encouraging therapeutic strategy for chemo-resistant breast cancers 26,27 .…”

PARP3 comes to light as a prime target in cancer therapy

“…Still, PARP inhibitors inhibit a family of proteins (17 members) with each protein having specific functions [3]. Among them, PARP3 has gained substantial attention in the field revealing unique biological properties in cell response to DNA damage, class-switch recombination, transcriptional regulation during neuronal development in the zebrafish, mitotic progression, and chromosome rearrangements [4][5][6][7][8][9][10].…”

Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage

“…Amplicons were sequenced by MiSeq (Illumina) at the CCIB DNA Core Facility at Massachusetts General Hospital (Cambridge, MA) using a MiSeq v2 chemistry 300 cycle kit. High-throughput analysis of amplicon deep sequencing was performed as in (79). Briefly, paired end sequencing raw reads were trimmed to primer sequences and merged into single reads using Geneious v10.1.3.…”