Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China.
University of Chinese Academy of Sciences, No.19A Yuquan Road, Beijing, 100049, China.
Cell Death Dis. 2020 Jan 28;11(1):71. doi: 10.1038/s41419-020-2265-y.
Poly(ADP-ribose) polymerase 1 (PARP1) regulates gene transcription in addition to functioning as a DNA repair factor. Forkhead box O1 (FoxO1) is a transcription factor involved in extensive biological processes. Here, we report that PARP1 binds to two separate motifs on the FoxO1 promoter and represses its transcription in a polymerase-independent manner. Using PARP1-knock out (KO) cells, wild-type-PARP1-complemented cells and catalytic mutant PARP1-reconstituted cells, we investigated transcriptional regulation by PARP1. PARP1 loss led to reduced DNA damage response and ~362-fold resistance to five PARP inhibitors (PARPis) in Ewing sarcoma cells. RNA sequencing showed 492 differentially expressed genes in a PARP1-KO subline, in which the FoxO1 mRNA levels increased up to more than five times. The change in the FoxO1 expression was confirmed at both mRNA and protein levels in different PARP1-KO and complemented cells. Moreover, exogenous PARP1 overexpression reduced the endogenous FoxO1 protein in RD-ES cells. Competitive EMSA and ChIP assays revealed that PARP1 specifically bound to the FoxO1 promoter. DNase I footprinting, mutation analyses, and DNA pulldown FREP assays showed that PARP1 bound to two particular nucleotide sequences separately located at -813 to -826 bp and -1805 to -1828 bp regions on the FoxO1 promoter. Either the PARPi olaparib or the PARP1 catalytic mutation (E988K) did not impair the repression of PARP1 on the FoxO1 expression. Exogenous FoxO1 overexpression did not impair cellular PARPi sensitivity. These findings demonstrate a new PARP1-gene promoter binding mode and a new transcriptional FoxO1 gene repressor.
聚（ADP-核糖）聚合酶 1（PARP1）除了作为 DNA 修复因子外，还调节基因转录。叉头框 O1（FoxO1）是一种参与广泛生物过程的转录因子。在这里，我们报告 PARP1 结合到 FoxO1 启动子上的两个独立基序上，并以聚合酶非依赖性方式抑制其转录。使用 PARP1 敲除（KO）细胞、野生型 PARP1 互补细胞和催化突变 PARP1 重建细胞，我们研究了 PARP1 的转录调节。PARP1 缺失导致 DNA 损伤反应减少，Ewing 肉瘤细胞对五种 PARP 抑制剂（PARPis）的抗性降低约 362 倍。RNA 测序显示 PARP1-KO 亚系中有 492 个差异表达基因，其中 FoxO1 mRNA 水平增加了超过五倍。在不同的 PARP1-KO 和互补细胞中，FoxO1 表达的变化在 mRNA 和蛋白质水平上均得到了证实。此外，外源性 PARP1 过表达降低了 RD-ES 细胞中的内源性 FoxO1 蛋白。竞争 EMSA 和 ChIP 测定显示 PARP1 特异性结合到 FoxO1 启动子上。DNase I 足迹分析、突变分析和 DNA 下拉 FREP 测定表明，PARP1 分别结合到 FoxO1 启动子上两个单独的核苷酸序列上，分别位于-813 到-826 bp 和-1805 到-1828 bp 区域。PARPi 奥拉帕利或 PARP1 催化突变（E988K）均不会损害 PARP1 对 FoxO1 表达的抑制作用。外源性 FoxO1 过表达不会损害细胞对 PARPi 的敏感性。这些发现表明了一种新的 PARP1-基因启动子结合模式和一种新的转录 FoxO1 基因抑制剂。
