RESUMO
USP7 is a deubiquitinase that regulates many diverse cellular processes, including tumor suppression, epigenetics, and genome stability. Several substrates, including GMPS, UHRF1, and ICP0, were shown to bear a specific KxxxK motif that interacts within the C-terminal region of USP7. We identified a similar motif in Enhancer of Zeste 2 (EZH2), the histone methyltransferase found within Polycomb Repressive Complex 2 (PRC2). PRC2 is responsible for the methylation of Histone 3 Lys27 (H3K27) leading to gene silencing. GST pull-down and coimmunoprecipitation experiments showed that USP7 interacts with EZH2. We determined the structural basis of interaction between USP7 and EZH2 and identified residues mediating the interaction. Mutations in these critical residues disrupted the interaction between USP7 and EZH2. Furthermore, USP7 silencing and knockout experiments showed decreased EZH2 levels in HCT116 carcinoma cells. Finally, we demonstrated decreased H3K27Me3 levels in HCT116 USP7 knockout cells. These results indicate that USP7 interacts with EZH2 and regulates both its stability and function.
Assuntos
Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Linhagem Celular Tumoral , Eletroforese em Gel de Poliacrilamida , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Inativação Gênica/fisiologia , Células HCT116 , Humanos , Imunoprecipitação , Complexo Repressor Polycomb 2/genética , Estabilidade Proteica , Peptidase 7 Específica de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/fisiologiaRESUMO
The minichromosome maintenance (MCM) complex, which plays multiple important roles in DNA replication, is loaded onto chromatin following mitosis, remains on chromatin until the completion of DNA synthesis, and then is unloaded by a poorly defined mechanism that involves the MCM binding protein (MCM-BP). Here we show that MCM-BP directly interacts with the ubiquitin-specific protease USP7, that this interaction occurs predominantly on chromatin, and that MCM-BP can tether USP7 to MCM proteins. Detailed biochemical and structure analyses of the USP7-MCM-BP interaction showed that the (155)PSTS(158) MCM-BP sequence mediates critical interactions with the TRAF domain binding pocket of USP7. Analysis of the effects of USP7 knockout on DNA replication revealed that lack of USP7 results in slowed progression through late S phase without globally affecting the fork rate or origin usage. Lack of USP7 also resulted in increased levels of MCM proteins on chromatin, and investigation of the cause of this increase revealed a defect in the dissociation of MCM proteins from chromatin in mid- to late S phase. This role of USP7 mirrors the previously described role for MCM-BP in MCM complex unloading and suggests that USP7 works with MCM-BP to unload MCM complexes from chromatin at the end of S phase.