ZNF432 stimulates PARylation and inhibits DNA resection to balance PARPi sensitivity and resistance.

Zinc finger (ZNF) motifs are some of the most frequently occurring domains in the human genome. It was only recently that ZNF proteins emerged as key regulators of genome integrity in mammalian cells. In this study, we report a new role for the Krüppel-type ZNF-containing protein ZNF432 as a novel poly(ADP-ribose) (PAR) reader that regulates the DNA damage response. We show that ZNF432 is recruited to DNA lesions via DNA- and PAR-dependent mechanisms. Remarkably, ZNF432 stimulates PARP-1 activity in vitro and in cellulo. Knockdown of ZNF432 inhibits phospho-DNA-PKcs and increases RAD51 foci formation following irradiation. Moreover, purified ZNF432 preferentially binds single-stranded DNA and impairs EXO1-mediated DNA resection. Consequently, the loss of ZNF432 in a cellular system leads to resistance to PARP inhibitors while its overexpression results in sensitivity. Taken together, our results support the emerging concept that ZNF-containing proteins can modulate PARylation, which can be embodied by the pivotal role of ZNF432 to finely balance the outcome of PARPi response by regulating homologous recombination.

The dynamic process of covalent and non-covalent PARylation in the maintenance of genome integrity: a focus on PARP inhibitors.

Poly(ADP-ribosylation) (PARylation) by poly(ADP-ribose) polymerases (PARPs) is a highly regulated process that consists of the covalent addition of polymers of ADP-ribose (PAR) through post-translational modifications of substrate proteins or non-covalent interactions with PAR via PAR binding domains and motifs, thereby reprogramming their functions. This modification is particularly known for its central role in the maintenance of genomic stability. However, how genomic integrity is controlled by an intricate interplay of covalent PARylation and non-covalent PAR binding remains largely unknown. Of importance, PARylation has caught recent attention for providing a mechanistic basis of synthetic lethality involving PARP inhibitors (PARPi), most notably in homologous recombination (HR)-deficient breast and ovarian tumors. The molecular mechanisms responsible for the anti-cancer effect of PARPi are thought to implicate both catalytic inhibition and trapping of PARP enzymes on DNA. However, the relative contribution of each on tumor-specific cytotoxicity is still unclear. It is paramount to understand these PAR-dependent mechanisms, given that resistance to PARPi is a challenge in the clinic. Deciphering the complex interplay between covalent PARylation and non-covalent PAR binding and defining how PARP trapping and non-trapping events contribute to PARPi anti-tumour activity is essential for developing improved therapeutic strategies. With this perspective, we review the current understanding of PARylation biology in the context of the DNA damage response (DDR) and the mechanisms underlying PARPi activity and resistance.

Functional Assessment of Coding and Regulatory Variants From the DKK1 Locus.

The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.