Lysine 27 of replication-independent histone H3.3 is required for Polycomb target gene silencing but not for gene activation

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.

细胞命运的精准判定，依赖于用于留存发育早期决策记忆的表观遗传（epigenetic）信息。组蛋白残基的翻译后修饰，被认为是表观遗传信息传递的核心途径。其中，组蛋白H3赖氨酸27（histone H3 lysine 27，H3K27）的修饰与基因激活和基因沉默均存在强相关性：H3K27乙酰化修饰存在于活跃转录位点，而H3K27甲基化修饰则分布于被多梳蛋白家族（Polycomb group proteins）沉默的基因座。携带这类修饰的组蛋白由复制依赖型H3基因与复制非依赖型H3.3基因共同编码。由于核小体周转速率存在差异，活跃基因座处的复制非依赖型H3.3组蛋白上富集了H3K27乙酰化修饰，而沉默基因座处的复制依赖型H3组蛋白则富集了H3K27甲基化修饰。既往研究表明，复制依赖型H3K27修饰对于多梳蛋白靶基因的沉默是必需的，但并非基因激活所必需。然而，复制非依赖型H3.3K27修饰在上述过程中的作用仍未明确。本研究利用CRISPR/Cas9技术，将内源性复制非依赖型H3.3K27位点突变为无法被修饰的氨基酸残基。令人意外的是，尽管H3.3与活跃转录相关，H3.3K27修饰对于多梳蛋白靶基因的沉默同样不可或缺。不过，H3.3K27的需求出现于发育的较晚阶段，晚于复制依赖型H3K27修饰的需求阶段，这提示有丝分裂后细胞对复制非依赖型H3.3K27修饰的依赖性更强。值得注意的是，尽管H3K27乙酰化与活跃转录存在强相关性，但H3.3K27突变体并未出现全局转录缺陷。