Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040, China; Institute of Urology, Fudan University, Shanghai 200040, China.
Department of Urology, Huashan Hospital, Fudan University, Shanghai 200040, China; Institute of Urology, Fudan University, Shanghai 200040, China.
Biochim Biophys Acta Gen Subj. 2023 Sep;1867(9):130417. doi: 10.1016/j.bbagen.2023.130417. Epub 2023 Jun 24.
Epigenetic mechanisms, including histone and DNA methylation, play a key role in kidney fibrosis, but the precise mechanism remains unclear. Concerted action between histone and DNA-methyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We investigated the role of G9a and DNMT1 in kidney fibrosis pathogenesis and aimed to elucidate key G9a and DNMT1 targets contributing to kidney fibrosis maintenance.
G9a and DNMT1 were detected in human fibrotic kidneys, UUO mouse kidneys, and TGFβ1-induced HK-2 cells. G9a and DNMT1 expression was knocked down by siRNA or inhibited with CM272 in HK-2 and UUO mouse, and transcriptomic responses to CM272 were examined. Antifibrogenic activity and safety of CM272 were studied in UUO mouse. Cell cycle were analyzed with flow cytometry. Gene expression regulation was analyzed by chromatin immunoprecipitation and methylation-specific PCR.
G9a and DNMT1 were overexpressed in human fibrotic kidneys, UUO mouse kidneys, and TGFβ1-induced HK-2 cells. G9a/DNMT1 inhibition potently alleviated fibrosis in vitro and vivo. G9a/DNMT1 inhibition reduced the expression of E2F targets and altered the methylation status of CDKN1A leading to the attenuated cell-cycle arrest. TGFβ1-induced overexpression of G9a or DNMT1 resulted in the enrichment of H3K9me2 and 5-methylcytosine at CDKN1A promoter.
Our data link G9a and DNMT1 to CDKN1A regulatory function and kidney fibrosis. Combined targeting G9a and DNMT1 could be a promising strategy for the treatment of kidney fibrosis.
表观遗传机制，包括组蛋白和 DNA 甲基化，在肾脏纤维化中发挥关键作用，但确切机制尚不清楚。组蛋白和 DNA 甲基转移酶（如 G9a 和 DNMT1）之间的协同作用是基因表达调控的一个共同主题。我们研究了 G9a 和 DNMT1 在肾脏纤维化发病机制中的作用，并旨在阐明对维持肾脏纤维化有贡献的关键 G9a 和 DNMT1 靶标。
检测了人类纤维化肾脏、UUO 小鼠肾脏和 TGFβ1 诱导的 HK-2 细胞中的 G9a 和 DNMT1。通过 siRNA 或 CM272 抑制 HK-2 和 UUO 小鼠中的 G9a 和 DNMT1 表达，并检测 CM272 的转录组反应。在 UUO 小鼠中研究了 CM272 的抗纤维化活性和安全性。用流式细胞术分析细胞周期。通过染色质免疫沉淀和甲基化特异性 PCR 分析基因表达调控。
G9a 和 DNMT1 在人类纤维化肾脏、UUO 小鼠肾脏和 TGFβ1 诱导的 HK-2 细胞中过度表达。G9a/DNMT1 抑制在体外和体内均能有效缓解纤维化。G9a/DNMT1 抑制降低了 E2F 靶基因的表达，并改变了 CDKN1A 的甲基化状态，导致细胞周期阻滞减弱。TGFβ1 诱导的 G9a 或 DNMT1 过表达导致 CDKN1A 启动子处 H3K9me2 和 5-甲基胞嘧啶的富集。
我们的数据将 G9a 和 DNMT1 与 CDKN1A 的调节功能和肾脏纤维化联系起来。联合靶向 G9a 和 DNMT1 可能是治疗肾脏纤维化的一种有前途的策略。
