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Nature
2013 Oct 10;5027470:249-53. doi: 10.1038/nature12488.
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Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication.
Nishiyama A
,
Yamaguchi L
,
Sharif J
,
Johmura Y
,
Kawamura T
,
Nakanishi K
,
Shimamura S
,
Arita K
,
Kodama T
,
Ishikawa F
,
Koseki H
,
Nakanishi M
.
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Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.
Figure 1: xUhrf1- and DNA-replication-dependent DNA methylation and ubiquitylation of H3 at lysine 23 in Xenopus egg extracts. a, Sperm chromatin was incubated with interphase egg extracts containing radiolabelled S-[methyl-3H]-adenosyl-L-methionine. His6–geminin, glutathione S-transferase–p27 or aphidicolin (APD, 150 µM) was added to the extracts 10 min before the addition of sperm DNA. After 1 h, sperm DNA was purified from the egg extracts and the incorporation of radioactivity was measured using a scintillation counter. Data shown are representative of three independent experiments. b, Interphase egg extracts immunodepleted (ID) with the indicated antiserum were incubated with sperm nuclei. Extracts and sperm chromatin isolated at the indicated times were analysed by immunoblotting (IB) using the indicated antibodies. c, Chromatin fractions from interphase extracts immunodepleted with the indicated antibodies were isolated at the indicated times after sperm addition and analysed by immunoblotting. d, Chromatin fractions from the indicated immunodepleted extracts were solubilized by MNase and immunoprecipitated with anti-H3 antibodies after treatment with 1% SDS. The resultant immunoprecipitates were subjected to immunoblotting using the indicated antibodies. e, The electron transfer dissociation MS/MS spectrum of the xH3 peptide shows ubiquitylation at lysine 23. Detailed information is provided in Methods. f, Buffer alone (−), recombinant His8-tagged wild-type (WT) rmDnmt1, or its catalytic mutant (cat) proteins (10 ng μl−1) were added to xDnmt1-depleted extracts. Chromatin fractions isolated at the indicated times were subjected to immunoblotting using the indicated antibodies.
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