XB-ART-53089Sci Rep. August 11, 2017; 7 (1): 55.
Usp7-dependent histone H3 deubiquitylation regulates maintenance of DNA methylation.
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.
PubMed ID: 28246399
PMC ID: PMC5427934
Article link: Sci Rep.
Genes referenced: dnmt1 pcna uhrf1 usp7
Article Images: [+] show captions
|Figure 2. Usp7 forms a stable complex with Dnmt1 in Xenopus egg extracts. (A) Dnmt1 was immunoprecipitated from interphase HSS using specific antibodies. The associated polypeptides were identified by mass spectrometry. The resultant immunoprecipitates were subjected to SDS-PAGE and stained with a SyproRuby; the peptides corresponding to Dnmt1 and Usp7 are indicated. The numbers of unique spectra identified for each protein are shown in the table. (B) Immunoprecipitates using anti-Dnmt1 (lane 3), anti-Usp7 (lane 4), anti-Uhrf1 (lane 5) antibodies, or control IgG (lane 2) as well as egg extracts (lane 1) were subjected to immunoblotting using the antibodies indicated. (C) Sequence alignment of the KG linker of Dnmt1 across different species. Red denotes residues mutated in a 4KA mutant used in this study. (D) FLAG-tagged wild-type or a 4KA mutant of recombinant Xenopus Dnmt1 was introduced into Xenopus egg extracts. Recombinant or endogenous Usp7 was immunoprecipitated using either anti-FLAG-M2 agarose beads or protein A agarose beads conjugated with anti-Usp7 antibodies, respectively. Immunoprecipitates were then subjected to immunoblotting using the antibodies indicated. Source data are available online for this figure.|
|Figure 7. A schematic model showing the role of Usp7 in the maintenance of DNA methylation. During S-phase, Uhrf1 binds to hemi-methylated DNA and ubiquitylates histone H3. Ubiquitylation of histone H3 facilitates the recruitment of Dnmt1/Usp7 complex to DNA methylation sites. Usp7 then deubiquitylates ubiquitylated histone H3, promoting DNA methylation.|