ZNF524 directly binds telomeres and supports their integrity via TRF2/RAP1

Telomeres are nucleoprotein structures at the ends of linear chromosomes. In humans, they consist of TTAGGG repeats, which are bound by dedicated proteins such as the shelterin complex. This complex blocks unwanted DNA damage repair at telomeres, e.g. by suppressing non-homologous end joining (NHEJ) through its subunit TRF2. We here describe ZNF524, a zinc finger protein that directly binds telomeric repeats with nanomolar affinity and reveal the base-specific sequence recognition by co-crystallization with telomeric DNA. ZNF524 localizes to telomeres in vivo, as shown among other approaches by ChIP-seq analysis, and specifically maintains the presence of the TRF2/RAP1 subcomplex at telomeres without affecting the other shelterin members. Loss of ZNF524 concomitantly results in an increase in DNA damage signaling and recombination events. Overall, ZNF524 is a direct telomere-binding protein involved in the maintenance of telomere integrity. Overall design: U2OS stable cell lines carrying ZNF524 WT-GFP ZNF524 ZF2 mutant-GFP or NLS-GFP were generated by lentiviral transduction followed by antibiotic selection and FACS sorting using the doxycycline-inducible pLIX_403 plasmid (Addgene #41395). Cells were seeded in medium supplemented with 300 ng/mL doxycycline 48h prior to the experiment to induce expression. GFP-Trap magnetic agarose beads (Chromotek) were used in these ChIP-seq reactions that were carried out in triplicates.

端粒（Telomeres）是线性染色体末端的核蛋白结构。在人类中，端粒由TTAGGG重复序列组成，并结合有专用调控蛋白，例如shelterin复合物（shelterin complex）。该复合物可阻断端粒处异常的DNA损伤修复，例如通过其亚基TRF2抑制非同源末端连接（non-homologous end joining, NHEJ）。本研究报道了ZNF524：一种能够以纳摩尔级亲和力直接结合端粒重复序列的锌指蛋白，并通过与端粒DNA共结晶的方式，揭示了其碱基特异性序列识别机制。实验证实，ZNF524在体内定位于端粒，除其他验证手段外，染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）分析也支持这一结论；同时，ZNF524可特异性维持TRF2/RAP1亚复合物在端粒处的存在，且不会影响其他shelterin复合物成员。ZNF524的缺失会伴随DNA损伤信号与重组事件的增加。综上，ZNF524是一种直接结合端粒的蛋白，参与端粒完整性的维持。总体实验设计：通过慢病毒转导，采用多西环素诱导型pLIX_403质粒（Addgene #41395）构建携带ZNF524野生型-绿色荧光蛋白（WT-GFP）、ZNF524 ZF2突变体-GFP或核定位信号-GFP（NLS-GFP）的U2OS稳定细胞系，随后经抗生素筛选与荧光激活细胞分选（fluorescence-activated cell sorting, FACS）完成纯化。实验前48小时，将细胞接种于添加300 ng/mL多西环素的培养基中以诱导靶蛋白表达。本研究的ChIP-seq实验采用GFP-Trap磁珠（Chromotek），并设置三次生物学重复。