M phase phosphorylation of the epigenetic regulator UHRF1 regulates its physical association with the deubiquitylase USP7 and stability

Abstract

UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) plays an important role in DNA CpG methylation, heterochromatin function and gene expression. Overexpression of UHRF1 has been suggested to contribute to tumorigenesis. However, regulation of UHRF1 is largely unknown. Here we show that the deubiquitylase USP7 interacts with UHRF1. Using interaction-defective and catalytic mutants of USP7 for complementation experiments, we demonstrate that both physical interaction and catalytic activity of USP7 are necessary for UHRF1 ubiquitylation and stability regulation. Mass spectrometry analysis identified phosphorylation of serine (S) 652 within the USP7-interacting domain of UHRF1, which was further confirmed by a UHRF1 S652 phosphor (S652ph)-specific antibody. Importantly, the S652ph antibody identifies phosphorylated UHRF1 in mitotic cells and consistently S652 can be phosphorylated by the M phase-specific kinase CDK1-cyclin B in vitro. UHRF1 S652 phosphorylation significantly reduces UHRF1 interaction with USP7 in vitro and in vivo, which is correlated with a decreased UHRF1 stability in the M phase of the cell cycle. In contrast, UHRF1 carrying the S652A mutation, which renders UHRF1 resistant to phosphorylation at S652, is more stable. Importantly, cells carrying the S652A mutant grow more slowly suggesting that maintaining an appropriate level of UHRF1 is important for cell proliferation regulation. Taken together, our findings uncovered a cell cycle-specific signaling event that relieves UHRF1 from its interaction with USP7, thus exposing UHRF1 to proteasome-mediated degradation. These findings identify a molecular mechanism by which cellular UHRF1 level is regulated, which may impact cell proliferation.deubiquitination | phosphorylation UHRF1 S652 E pigenetic regulation has emerged as an important mechanism that regulates many chromatin template-based processes, including transcription, DNA replication, and repair. An important component of epigenetic regulation is DNA CpG methylation, which is mediated by DNA methyltransferases such as DMNT1 and DNMT3a/b and an accessory factor DNMT3L (1, 2). Recent studies demonstrate that maintenance of DNA methylation patterns requires UHRF1 (Ubiquitin-like, with PHD and RING finger domains 1) (also called Np95 and ICBP90). UHRF1 binds hemimethylated CpG and recruits DNMT1 to ensure faithful propagation of the DNA methylation patterns through DNA replication (3, 4). UHRF1 is also localized to euchromatic regions where it regulates transcription possibly by impacting DNA methylation and histone modifications (5, 6). UHRF1 has been shown to regulate cell proliferation, and its loss has been implicated in the mis-regulation of both G1 and G2/M phases of the cell cycle, respectively (7). However, very little is known how this important epigenetic regulator itself is regulated. To address this question, we have recently undertaken a proteomics approach and identified a cell cycle signaling-regulated physical interaction of UHRF1 with the deubiquitylase USP7 (HAUSP) (8,9) ...

Deubiquitination of Tip60 by USP7 Determines the Activity of the p53-Dependent Apoptotic Pathway

“…7G). Alternatively, the deubiquitination activity of USP7 is also required for the stability of other proteins such as PTEN, Claspin, FOXO4, UHRF1, and NF-B (42)(43)(44)(45)(46). It is therefore also possible that the residual cytotoxicity of P22077 in cells overexpressing Flag-Tip60 is due to a decrease of the levels of these other cellular targets of USP7.…”

DNA Damage Regulates UHRF1 Stability via the SCFβ-TrCP E3 Ligase

“…At the M phase of the cell cycle, USP7 disassociates from UHRF1, thus exposing UHRF1 to proteasomal degradation (18). Importantly, manipulating the UHRF1 level in cells has been shown to affect cell proliferation (11,18,20). Collectively, these findings suggest that maintaining an appropriate level of UHRF1 is important for processes such as cell proliferation regulation and the DDR.…”

“…More recent studies suggest that UHRF1 turnover is controlled by proteasome-mediated degradation. These studies identified the deubiquitylase USP7 in the regulation of the UHRF1 level in vivo (17)(18)(19). Specifically, UHRF1 is protected from proteasome-mediated degradation through its association with the deubiquitylase USP7, in a cell cycle-dependent manner.…”

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“…Alterations in DNA methylation status are associated with many biological processes, including wound healing and fibrosis, [29][30][31][32] DNA repair, 33,34 cell cycle regulation, [35][36] inflammatory/stress response, 37,38 apoptosis, and tumorigenesis. 39 DNA methylation at the 5 position of cytosine within CpG dinucleotides epigenetically controls gene expression and maintains genome integrity.…”