Center for Epigenetics, Van Andel Research Institute, Grand Rapids, MI 49503.
EpiCypher, Inc., Research Triangle Park, NC 27713.
Proc Natl Acad Sci U S A. 2018 Aug 28;115(35):8775-8780. doi: 10.1073/pnas.1806373115. Epub 2018 Aug 13.
Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.
UHRF1 促进 DNA 甲基化模式的有丝分裂遗传，它是一种 DNA 和组蛋白结合的 E3 泛素连接酶，有助于招募维持性 DNA 甲基转移酶 DNMT1 到复制染色质。UHRF1 的 DNA 甲基化维持功能依赖于其与染色质结合的能力，在染色质中，它促进组蛋白 H3 在赖氨酸 18 和 23 处的单泛素化，这是 DNMT1 的一个对接位点。由于技术限制，这种依赖 UHRF1 的 DNA 甲基化遗传模型主要是基于遗传学和生物化学观察构建的，这些观察涉及甲基化 DNA 寡核苷酸、合成组蛋白肽和从细胞中提取的异质染色质。在这里，我们构建了含有定义明确的组蛋白和 DNA 修饰的半合成单核小体，并使用这些试剂对 UHRF1 结合和酶活性进行了严格的分析。我们表明，核小体连接 DNA 的多价结合和组蛋白 H3 上的二甲基化赖氨酸 9 指导 UHRF1 泛素连接酶活性朝向组蛋白底物。值得注意的是，我们揭示了一个分子开关，由对半甲基化 DNA 的识别刺激，它将 UHRF1 泛素连接酶活性从组蛋白上重新定向，有利于强大的自泛素化。我们的研究支持 UHRF1 和 DNMT1 向复制染色质募集的非竞争性模型，并定义了半甲基化连接 DNA 作为 UHRF1 泛素连接酶底物选择性调节剂的作用。
