Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.
PLoS Genet. 2019 Jan 30;15(1):e1007932. doi: 10.1371/journal.pgen.1007932. eCollection 2019 Jan.
Proper determination of cell fates depends on epigenetic information that is used to preserve memory of decisions made earlier in development. Post-translational modification of histone residues is thought to be a central means by which epigenetic information is propagated. In particular, modifications of histone H3 lysine 27 (H3K27) are strongly correlated with both gene activation and gene repression. H3K27 acetylation is found at sites of active transcription, whereas H3K27 methylation is found at loci silenced by Polycomb group proteins. The histones bearing these modifications are encoded by the replication-dependent H3 genes as well as the replication-independent H3.3 genes. Owing to differential rates of nucleosome turnover, H3K27 acetylation is enriched on replication-independent H3.3 histones at active gene loci, and H3K27 methylation is enriched on replication-dependent H3 histones across silenced gene loci. Previously, we found that modification of replication-dependent H3K27 is required for Polycomb target gene silencing, but it is not required for gene activation. However, the contribution of replication-independent H3.3K27 to these functions is unknown. Here, we used CRISPR/Cas9 to mutate the endogenous replication-independent H3.3K27 to a non-modifiable residue. Surprisingly, we find that H3.3K27 is also required for Polycomb target gene silencing despite the association of H3.3 with active transcription. However, the requirement for H3.3K27 comes at a later stage of development than that found for replication-dependent H3K27, suggesting a greater reliance on replication-independent H3.3K27 in post-mitotic cells. Notably, we find no evidence of global transcriptional defects in H3.3K27 mutants, despite the strong correlation between H3.3K27 acetylation and active transcription.
正确的细胞命运决定取决于表观遗传信息，这些信息被用来保存发育早期所做决定的记忆。组蛋白残基的翻译后修饰被认为是传播表观遗传信息的核心手段。特别是，组蛋白 H3 赖氨酸 27（H3K27）的修饰与基因激活和基因抑制都密切相关。H3K27 乙酰化发生在活跃转录的位点，而 H3K27 甲基化发生在被 Polycomb 组蛋白沉默的基因座。带有这些修饰的组蛋白由依赖复制的 H3 基因以及非依赖复制的 H3.3 基因编码。由于核小体周转率的差异，H3K27 乙酰化在活跃基因座的非依赖复制的 H3.3 组蛋白上富集，而 H3K27 甲基化在沉默基因座的依赖复制的 H3 组蛋白上富集。此前，我们发现依赖复制的 H3K27 修饰对于 Polycomb 靶基因沉默是必需的，但对于基因激活则不是必需的。然而，非依赖复制的 H3.3K27 对这些功能的贡献尚不清楚。在这里，我们使用 CRISPR/Cas9 技术将内源性非依赖复制的 H3.3K27 突变为不可修饰的残基。令人惊讶的是，我们发现尽管 H3.3 与活跃转录有关，但 H3.3K27 对于 Polycomb 靶基因沉默也是必需的。然而，H3.3K27 的需求出现在比依赖复制的 H3K27 更晚的发育阶段，这表明在有丝分裂后的细胞中，非依赖复制的 H3.3K27 的依赖性更大。值得注意的是，尽管 H3.3K27 乙酰化与活跃转录之间存在很强的相关性，但我们没有发现 H3.3K27 突变体存在全局转录缺陷的证据。
