Department of Biochemistry III, Universität Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany.
Nucleic Acids Res. 2011 Oct;39(19):8355-65. doi: 10.1093/nar/gkr528. Epub 2011 Jul 10.
Aberrant DNA methylation is often associated with cancer and the formation of tumors; however, the underlying mechanisms, in particular the recruitment and regulation of DNA methyltransferases remain largely unknown. In this study, we identified USP7 as an interaction partner of Dnmt1 and UHRF1 in vivo. Dnmt1 and USP7 formed a soluble dimer complex that associated with UHRF1 as a trimeric complex on chromatin. Complex interactions were mediated by the C-terminal domain of USP7 with the TS-domain of Dnmt1, whereas the TRAF-domain of USP7 bound to the SRA-domain of UHRF1. USP7 was capable of targeting UHRF1 for deubiquitination and affects UHRF1 protein stability in vivo. Furthermore, Dnmt1, UHRF1 and USP7 co-localized on silenced, methylated genes in vivo. Strikingly, when analyzing the impact of UHRF1 and USP7 on Dnmt1-dependent DNA methylation, we found that USP7 stimulated both the maintenance and de novo DNA methylation activity of Dnmt1 in vitro. Therefore, we propose a dual role of USP7, regulating the protein turnover of UHRF1 and stimulating the enzymatic activity of Dnmt1 in vitro and in vivo.
异常的 DNA 甲基化通常与癌症和肿瘤的形成有关；然而，其潜在机制，特别是 DNA 甲基转移酶的募集和调节在很大程度上仍是未知的。在这项研究中，我们鉴定了 USP7 是 Dnmt1 和 UHRF1 的体内相互作用伙伴。Dnmt1 和 USP7 形成可溶的二聚体复合物，该复合物与 UHRF1 作为三聚体复合物在染色质上结合。复合物相互作用是由 USP7 的 C 末端结构域与 Dnmt1 的 TS 结构域介导的，而 USP7 的 TRAF 结构域与 UHRF1 的 SRA 结构域结合。USP7 能够靶向 UHRF1 进行去泛素化，并影响体内 UHRF1 蛋白的稳定性。此外，Dnmt1、UHRF1 和 USP7 在体内沉默的、甲基化的基因上共定位。引人注目的是，当分析 UHRF1 和 USP7 对 Dnmt1 依赖性 DNA 甲基化的影响时，我们发现 USP7 刺激了 Dnmt1 在体外的维持和从头 DNA 甲基化活性。因此，我们提出了 USP7 的双重作用，调节 UHRF1 的蛋白周转和刺激 Dnmt1 在体外和体内的酶活性。
