Abstract B45: A genome-scale screen identifies the microcephaly gene, ZNF335, as a regulator of DNA end resection

Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair pathway that is of critical importance during the S and G2 phases of the mammalian cell cycle. The limiting step in the HR pathway is the generation of single-stranded DNA (ssDNA) by resection of DNA ends at the DSB site. We have established a high-throughput immunofluorescence-based assay that monitors DNA end resection by plate-based laser scanning cytometry. Employing this assay we screened a genome-scale siRNA library that targeted approximately 18,500 genes. Remarkably, the top hits in our screen were known regulators of resection including CtIP and all three subunits of the MRN complex. I will present ongoing work on one of the hits from the screen, the zinc finger protein ZNF335. The ZNF335 gene was recently found to be mutated in a syndrome that causes some of the worst cases of neonatal microcephaly ever observed. Numerous genome instability syndromes have been documented to display microcephaly as a clinical feature, including Seckel syndrome, which can be caused by mutations in the known resection genes CtIP and DNA2 . We first validated the results of the screen by demonstrating that ZNF335 promotes resection and repair by HR. We determined that the four C-terminal zinc finger domains of ZNF335 are necessary and sufficient for its function in resection. Depletion of ZNF335 inhibits phosphorylation of CHK1 at a characterized ATR consensus site, suggesting that ZNF335 is needed for activation of ATR following a DSB. We examined the recruitment of known resection factors to DSB sites and determined that the localization of CtIP and BLM were impaired in cells depleted of ZNF335. Our data suggests that ZNF335 plays an important role in promoting resection and ATR activation in response to DSBs. Citation Format: Jordan Young, Mikhail Bashkurov, Andrea McEwan, Thomas Sun, Alessandro Datti, Daniel Durocher. A genome-scale screen identifies the microcephaly gene, ZNF335, as a regulator of DNA end resection [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B45.