ATM mediates pRB function to control DNMT1 protein stability and DNA methylation. Mus musculus

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many of chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability, and hence controls the DNA methylation status in the promoter of at least Ink4a, Shc2, FoxO6 and Noggin genes. Further, we demonstrate that inactivation of pRB promotes the Tip60 (acetyltransferase)-dependent ATM activation, allows activated ATM to physically bind to DNMT1 forming a complex with Tip60 and UHRF1 (E3 ligase), and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of pRB pathway in coordination with aberration in DNA damage response leads to abnormal DNA methylation pattern by affecting the stability of DNMT1. Overall design: 163; Rb-/-;ATM-/- MEFs, D4; DNMT1 knockdown from 163 cells and analysed at day 4, WT8S; 163 cells reconstituted with ATM expression vector

视网膜母细胞瘤抑癌基因（RB）的编码产物可通过物理结合多种染色质修饰因子，参与基因表达的表观遗传调控。然而，该功能的生物学与临床意义尚未得到充分阐明。为解答这一科学问题，我们在RB缺陷型小鼠甲状腺C细胞瘤模型中开展了遗传与表观遗传分析。本研究报道，RB与ATM（毛细血管扩张性共济失调突变激酶）的遗传互作可调控DNA甲基转移酶1（DNMT1）的蛋白质稳定性，进而调控至少Ink4a、Shc2、FoxO6及Noggin基因启动子区域的DNA甲基化状态。进一步研究表明，pRB失活会促进Tip60（乙酰转移酶）依赖的ATM激活，使激活的ATM能够与DNMT1发生物理结合，并与Tip60和UHRF1（E3连接酶）形成复合物，最终加速由Tip60依赖的乙酰化介导的DNMT1泛素化。本研究结果显示，pRB通路失活配合DNA损伤应答异常，可通过影响DNMT1的稳定性引发异常DNA甲基化模式。整体实验设计：163细胞、Rb基因敲除（Rb-/-）且ATM基因敲除（ATM-/-）的小鼠胚胎成纤维细胞（MEFs），培养至第4天（D4）；对163细胞进行DNMT1敲低，并于第4天开展分析；WT8S：通过ATM表达载体重构的163细胞。