Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation

Luna Yamaguchi,Chikashi Obuse,Toshinori Misaki,Koji Nagao,Makoto Nakanishi,Kyohei Arita,Atsuya Nishiyama,Jun Ueda,Yoshikazu Johmura

doi:10.1038/s41598-017-00136-5

Abstract

Uhrf1-dependent histone H3 ubiquitylation plays a crucial role in the maintenance of DNA methylation via the recruitment of the DNA methyltransferase Dnmt1 to DNA methylation sites. However, the involvement of deubiquitylating enzymes (DUBs) targeting ubiquitylated histone H3 in the maintenance of DNA methylation is largely unknown. With the use of Xenopus egg extracts, we demonstrate here that Usp7, a ubiquitin carboxyl-terminal hydrolase, forms a stable complex with Dnmt1 and is recruited to DNA methylation sites during DNA replication. Usp7 deubiquitylates ubiquitylated histone H3 in vitro. Inhibition of Usp7 activity or its depletion in egg extracts results in enhanced and extended binding of Dnmt1 to chromatin, suppressing DNA methylation. Depletion of Usp7 in HeLa cells causes enhanced histone H3 ubiquitylation and enlargement of Dnmt1 nuclear foci during DNA replication. Our results thus suggest that Usp7 is a key factor that regulates maintenance of DNA methylation.

Highlights

In higher eukaryotes, DNA methylation plays a crucial role in many biological processes, such as transcriptional regulation of developmental genes, silencing of transposable elements, and X-chromosome inactivation, and is involved in maintenance of genomic integrity and tumorigenesis[1,2,3,4]
Histone H3 ubiquitylation is critical for recruitment of Dnmt[1] to DNA replication sites, it is hardly detectable during normal S-phase
To examine whether certain deubiquitylating enzymes (DUBs)(s) are involved in this rapid process, we first examined the effect of ubiquitin-vinyl-sulfone (Ub-VS), a pan-inhibitor of only active DUBs25, on histone H3 ubiquitylation and maintenance DNA methylation

Summary

Introduction

DNA methylation plays a crucial role in many biological processes, such as transcriptional regulation of developmental genes, silencing of transposable elements, and X-chromosome inactivation, and is involved in maintenance of genomic integrity and tumorigenesis[1,2,3,4]. Dnmt[1] converts hemi-methylated DNA to fully-methylated DNA in a processive manner[12, 13] This conversion displaces both Uhrf[1] and Dnmt[1] from chromatin. We previously reported that interphase Xenopus egg extracts successfully recapitulate Uhrf1-dependent DNA methylation in vitro, and Uhrf1-dependent histone H3 ubiquitylation is necessary for Dnmt[1] recruitment to sites of DNA methylation[16]. Ubiquitylated histone H3 disappears when DNA replication is completed This ubiquitylation greatly accumulates when Dnmt[1] function is aberrant[16]. These results suggest that histone H3 ubiquitylation might regulate the dynamic action of Dnmt[1] during the maintenance of DNA methylation. Our results suggest that histone H3 deubiquitylation by Usp[7] is an important step in the maintenance of DNA methylation

Methods

Results

Conclusion