USP7 negatively controls global DNA methylation by attenuating ubiquitinated histone-dependent DNMT1 recruitment

Jialun Li,Xueli Hu,Weiyi Lai,Cong Lyu,Pishun Li,Huifang Gao,Zhaosu Chen,Kongbin Lu,Jiaxu Jin ,Yingying Gao,Hongxia Zhu ,Ruiping Wang,Guang Hu,Hailin Wang,Qihan Wu,Jiwen Li,Yanjiao Shao,Mengmeng Han,Dali Li,Jiemin Wong

doi:10.1038/s41421-020-00188-4

Abstract

Previous studies have implicated an essential role for UHRF1-mediated histone H3 ubiquitination in recruiting DNMT1 to replication sites for DNA maintenance methylation during S phase of the cell cycle. However, the regulatory mechanism on UHRF1-mediated histone ubiquitination is not clear. Here we present evidence that UHRF1 and USP7 oppositely control ubiquitination of histones H3 and H2B in S phase of the cell cycle and that DNMT1 binds both ubiquitinated H3 and H2B. USP7 knockout markedly increased the levels of ubiquitinated H3 and H2B in S phase, the association of DNMT1 with replication sites and importantly, led to a progressive increase of global DNA methylation shown with increased cell passages. Using DNMT3A/DNMT3B/USP7 triple knockout cells and various DNA methylation analyses, we demonstrated that USP7 knockout led to an overall elevation of DNA methylation levels. Mechanistic study demonstrated that USP7 suppresses DNMT1 recruitment and DNA methylation through its deubiquitinase activity and the interaction with DNMT1. Altogether our study provides evidence that USP7 is a negative regulator of global DNA methylation and that USP7 protects the genome from excessive DNA methylation by attenuating histone ubiquitination-dependent DNMT1 recruitment.

Highlights

In mammals, DNA methylation at cytosine is an epigenetic modification required for embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting, transposon silencing, and genome stability[1,2,3]
We ectopically overexpressed wild-type, a deubiquitinase-deficient USP7 C223S mutant (USP7m) (Fig. 1a)[32] and UHRF1 in Human embryonic kidney 293T cell line (HEK293T) cells and synchronized cells to S phase by aphidicolin treatment followed by a 4 h-release in fresh medium
Yarychkivska et al recently reported that knockout of USP7 in mouse embryonic fibroblasts (MEFs) and knockdown of USP7 in H1299 cells had no significant effect on the global levels of DNMT1 protein[45]

Summary

Introduction

DNA methylation at cytosine is an epigenetic modification required for embryonic development, transcriptional regulation, heterochromatin formation, X-inactivation, imprinting, transposon silencing, and genome stability[1,2,3]. Accumulative studies have established a key role for UHRF1 ( known as ICBP90 in humans and NP95 in mice) and its catalyzed histone H3 ubiquitination in targeting DNMT1 to DNA replication fork[11,12,13,14]. A recent study[29] provides evidence that UHRF1 can associate with replication fork by binding methylated DNA ligase 1. The initial work suggested that replication fork-associated UHRF1 may recruit DNMT1 via a direct protein–protein interaction[11,12], recent studies provide compelling evidence that UHRF1’s ubiquitin E3 ligase activity is required for DNMT1 recruitment[13,14] and ubiquitinated H3 binds DNMT1 and activates DNMT1 enzymatic activity[30]. UHRF1 has been shown to ubiquitinate H3 N-terminal tail at multiple sites[13,14,31], whereas

Methods

Results

Conclusion